Inhibition of hdac2 to promote memory

ABSTRACT

The invention relates to methods and products for enhancing and improving recovery of lost memories. In particular the methods are accomplished by inhibiting HDAC2 and or selectively inhibiting HDAC1/2 or HDAC1/2/3.

RELATED APPLICATION

This application claims priority under 35 USC §119 to U.S. ProvisionalApplication No. 61/119,698, filed Dec. 3, 2008, the entire contents ofwhich is hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NIH NS051874.Accordingly, the Government has certain rights in this invention.

BACKGROUND OF INVENTION

Brain atrophy occurs during normal aging and is an early feature ofneurodegenerative diseases associated with impaired learning and memory.Only recently have mouse models with extensive neurodegeneration in theforebrain been reported (1-3). One of these models is the bi-transgenicCK-p25 Tg mice where expression of p25, a protein implicated in variousneurodegenerative diseases (4), is under the control of the CamKIIpromoter and can be switched on or off with a doxycycline diet (3,5).Post-natal induction of p25 expression for 6 weeks caused learningimpairment that was accompanied by severe synaptic and neuronal loss inthe forebrain. However, pre-clinical research has not yet exploredstrategies to recover lost memories after substantial neuronal loss hadtaken place.

Neuronal adaptive responses, implicated in memory formation and storage,involve functional and structural synaptic changes, which requirealterations in gene expression (West, A. E. et al. Proc Natl Acad SciUSA 98 (20), 11024-11031 (2001); Guan, Z. et al. Cell 111 (4), 483-493(2002)). The mechanisms underlying this process are still unclear.Chromatin remodeling, especially through histone-tail acetylation, whichalters the compact chromatin structure and changes the accessibility ofDNA to regulatory proteins, is emerging as a fundamental mechanism forregulation of gene expression in development and adulthood (Kurdistani,S. K. & Grunstein, M. Nat Rev Mol Cell Biol 4 (4), 276-284 (2003);Goldberg, A. D., Allis, C. D., & Bernstein, E. Cell 128 (4), 635-638(2007)).

SUMMARY OF INVENTION

Neurodegenerative diseases of the central nervous system are oftenassociated with impaired learning and memory, eventually leading todementia. An important aspect that has not been addressed extensively inpre-clinical research, is the loss of long-term memories and theexploration of strategies to re-establish access to those memories. Insome embodiments the current invention provides methods for restoringaccess to long-term memory after synaptic and neuronal loss has alreadyoccurred. Environmental enrichment (EE) has been shown to reinstatelearning behavior and re-establish access to long-term memories aftersignificant brain atrophy and neuronal loss has already occurred. Alsoshown herein is a correlation between EE and epigenetic changes. EEincreases histone-tail acetylation and changes the level of methylation.The increase in acetylation and change in level of methylation isobserved in hippocampal and cortical histone 3 (H3) and histone 4 (H4).In turn, elevated histone H3 and H4 acetylation initiate rewiring of theneural network.

In some aspects the invention is a method for enhancing a memory in asubject by administering to the subject an HDAC2 inhibitor in an amounteffective to enhance the memory in the subject. The HDAC2 inhibitor maybe a selective HDAC2 inhibitor. In other embodiments the HDAC2 inhibitoris non-selective but is not an HDAC1, HDAC5, HDAC6, HDAC7 and/or HDAC10inhibitor. In yet other embodiments the HDAC2 inhibitor is anHDAC1/HDAC2 selective inhibitor or an HDAC1/HDAC2/HDAC3 selectiveinhibitor.

In some embodiments the invention provides a method for accessinglong-term memory in a subject having diminished access to a long-termmemory comprising increasing histone acetylation in an amount effectiveto reestablish access to long-term memory in the subject.

In some aspects of the invention the long-term memory is impaired. Insome embodiments the impairment may be age-related or injury-related. Insome embodiments of the invention a synaptic network in the subject isre-established. In some embodiments re-establishing the synaptic networkcomprises an increase in the number of active brain synapses. In someembodiments re-establishing the synaptic network comprises a reversal ofneuronal loss. In some embodiments the subject has a disorder selectedfrom the group consisting of MCI (mild cognitive impairment),Alzheimer's Disease, memory loss, attention deficit symptoms associatedwith Alzheimer disease, neurodegeneration associated with Alzheimerdisease, dementia of mixed vascular origin, dementia of degenerativeorigin, pre-senile dementia, senile dementia, dementia associated withParkinson's disease, vascular dementia, progressive supranuclear palsyor cortical basal degeneration.

The methods optionally involve administration of additional compounds.For instance, in some embodiments a HDAC3 inhibitor is administered. Inother embodiments a HDAC11 inhibitor is administered. In yet otherembodiments a DNA methylation inhibitor such as 5-azacytidine,5-aza-2′deoxycytidine, 5,6-dihydro-5-azacytidine,5,6-dihydro-5-aza-2′deoxycytidine, 5-fluorocytidine,5-fluoro-2′deoxycytidine, and short oligonucleotides containing5-aza-2′deoxycytosine, 5,6-dihydro-5-aza-2′deoxycytosine, and5-fluoro-2′deoxycytosine, and procainamide, Zebularine, and(−)-egallocatechin-3-gallate is administered. An additional therapeuticagent such as ARICEPT or donepezil, COGNEX or tacrine, EXELON orrivastigmine, REMINYL or galantamine, anti-amyloid vaccine,Abeta-lowering therapies, mental exercise or stimulation may beadministered.

In other embodiments the HDAC2 inhibitor is an HDAC2 RNAi such as asiRNA, shRNA, miRNA, dsRNA or ribozyme or variants thereof.

The HDAC2 inhibitor may be administered orally, intravenously,cutaneously, subcutaneously, nasally, imtramuscularly,intraperitoneally, intracranially, or intracerebroventricularly.

The methods may also include a step of assessing cognitive function ofthe subject after administration of the HDAC2 inhibitor. Further themethod may involve monitoring treatment by assessing cerebral blood flowor blood-brain barrier function.

A method for treating Alzheimer's disease by administering to a subjecthaving Alzheimer's disease an HDAC2 inhibitor in an amount effective totreat Alzheimer's disease is provided according to other aspects of theinvention. In one embodiment the HDAC2 inhibitor is a selective HDAC2inhibitor.

In some embodiments the HDAC2 inhibitor is a selective HDAC1/HDAC2inhibitor. In other embodiments the HDAC2 inhibitor is a selectiveHDAC1/HDAC2/HDAC3 inhibitor. In some embodiments, the HDAC2 inhibitor isa selective HDAC1/HDAC2/HDAC10 inhibitor. In some embodiments, theselective HDAC1/HDAC2/HDAC10 inhibitor is BRD-6929. In otherembodiments, the HDAC2 inhibitor is a selective HDAC1/HDAC2/HDAC3/HDAC10inhibitor.

In yet other embodiments the HDAC2 inhibitor is a compound of formula(IV)

wherein R₁ and R₂ are independently selected from H, and—C(O)—C₁₋₆alkyl; R₃ is optionally substituted aryl, optionallysubstituted heteroaryl, or aryl-C₁₋₆alkylene.

In some embodiments R₁ is H; R₁ and R₂ are H; R₁ is —C(O)—C₁₋₆alkyl; R₁is —C(O)-methyl; R₁ is —C(O)-methyl and R₂ is H; R₃ is optionallysubstituted aryl; R₃ is tolyl; R₃ is optionally substituted heteroaryl;R₃ is thienyl; R₃ is aryl-C₁₋₆alkylene; or R₃ is phenyl-ethylene.

In other embodiments formula IV is

In other embodiments formula IV is

In other embodiments formula IV is

In other embodiments formula IV is

The HDAC2 inhibitor in other embodiments is a compound of formula (VI)

wherein R₁ and R₂ are independently selected from H, substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₁₋₆alkyl,heterocyclyl, heteroaryl, aryl, and aryl-C₁₋₆alkylene.

In some embodiments R₁ is H; R₁ and R₂ are H: R₁ is methyl, ethyl,propyl, or butyl; R₁ is aryl-C₁₋₆alkylene; R₁ is phenyl-ethylene; or R₂is H.

In other embodiments formula VI is

The HDAC2 inhibitor in other embodiments is a compound of formula (I)

wherein R₁ and R₂ are independently selected from H, substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₁₋₆alkyl,heterocyclyl, C₁₋₆alkylene, heteroaryl, heteroarylene, andheteroarylene-alkylene; and R₃ is aryl or heteroaryl.

In some embodiments R₁ is unsubstituted acyclic C₁₋₆alkyl; R₁ isselected from a group consisting of methyl, ethyl, propyl, and butyl; R₁is heteroarylene-alkylene; R₁ is heteroarylene-C₁₋₆alkylene; R₁ ispyridinyl-ethylene; R₂ is hydrogen; R₃ is heteroaryl; or R₃ is thienyl.

In yet other embodiments formula I is

The HDAC2 inhibitor in some embodiments is a compound of formula (II)

wherein R₁ and R₂ are independently selected from H, substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₁₋₆alkyl,heterocyclyl, C₁₋₆alkylene, heteroaryl, heteroarylene,heteroarylene-alkylene, arylene-alkylene; and heterocyclyl-alkyleneoptionally substituted; and R₃ is aryl or heteroaryl.

In some embodiments R₁ is unsubstituted acyclic C₁₋₆alkyl; R₁ isselected from a group consisting of methyl, ethyl, propyl, and butyl; R₁is heteroarylene-alkylene; R₁ is heteroarylene-C₁₋₆alkylene; R₁ ispyridinyl-ethylene; R₁ is arylene-alkylene; R₁ is arylene-C₁₋₆alkylene;R₁ is phenyl-ethylene; R₁ is heterocyclyl-alkylene; R₁ is unsubstitutedheterocyclyl-C₁₋₆alkylene; R₁ is piperazine-ethylene; R₁ is substitutedheterocyclyl-C₁₋₆alkylene; R₁ is substituted piperazine-ethylene; R₁ isC₁₋₆alkylene substituted piperazine-ethylene; R₁ is methyl substitutedpiperazine-ethylene; R₂ is hydrogen; R₃ is heteroaryl; R₃ is thienyl; orR₃ is pyridinyl.

In other embodiments formula II is

In other embodiments formula II is

In other embodiments formula II is

In other embodiments formula II is

In other embodiments formula II is

The HDAC2 inhibitor in some embodiments is a compound of formula (III)

wherein X is —C(O)—N(R₁)(R₂),C₁₋₆alkylene-N(H)—C₁₋₆alkylene-N(R₁)C(O)(R₂); or —N(R₁)C(O)R₂; R₁ and R₂are independently selected from H, and substituted or unsubstituted,branched or unbranched, cyclic or acyclic C₁₋₆alkyl; and R₃ is alkynyl,aryl, or heteroaryl.

In some embodiments X is —C(O)—N(R₁)(R₂); R₁ and R₂ are independentlyselected from H, unsubstituted, unbranched, acyclic C₁₋₆alkyl; R₁ and R₂are independently selected from H, methyl, ethyl, propyl, and butyl; R₁is H; R₁ and R₂ are H; X is —C(O)—NH₂; X isC₁₋₆alkylene-N(R₁)—C₁₋₆alkylene-N(R₁)C(O)(R₂); R₁ is H; X isC₁₋₆alkylene-N(H)—C₁₋₆alkylene-N(H)C(O)(R₂); X is —N(R₁)C(O)R₂; R₁ is H;R₁ is unsubstituted acyclic C₁₋₆alkyl; R₁ is selected from a groupconsisting of methyl, ethyl, propyl, and butyl; R₂ is unsubstitutedacyclic C₁₋₆alky; R₂ is selected from a group consisting of methyl,ethyl, propyl, and butyl; R₃ is heteroaryl; R₃ is thienyl; R₃ is aryl;R₃ is alkynyl; R₃ is C₁₋₆alkynyl; or R₃ is ethynyl.

In some embodiments formula III is

In some embodiments formula III is

In some embodiments formula III is

The HDAC2 inhibitor in other embodiments is a compound of formula (V)

wherein R₁ and R₂ are independently selected from H, and substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₁₋₆alkyl; andR₃ is aryl or heteroaryl.

In some embodiments R₁ is H; R₁ and R₂ are H; R₁ is methyl, ethyl,propyl, or to butyl; R₃ is aryl; R₃ is heteroaryl; or R₃ is thienyl.

In other embodiments formula V is

In some embodiments the methods specifically exclude the use ofmolecules of Formula IV.

Pharmaceutical compositions of a HDAC2 inhibitor and a pharmaceuticallyacceptable carrier in a formulation for delivery to brain tissue arealso provided. In some embodiments the HDAC2 inhibitor is formulated forcrossing blood brain barrier.

In other aspects the invention is a composition of an HDAC2 inhibitor,wherein the HDAC2 inhibitor is selected from the group consisting ofcompounds of formula I, II and III.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 shows HDAC inhibitor improved associative learning via HDAC2. a.Memory test for mice with contextual fear conditioning training (footshock 1.0 mA). HDAC2OE mice (SAHA group, n=12; saline group, n=12) andWT littermates (SAHA group, n=12; saline group, n=15) were treated withsaline or SAHA (25 mg/kg, i.p.) for 10 days before memory test. b. CA1region (pyramidal neuron layer; stratum radiatum (s.r.)) from WT andHDAC2OE mice received chronic SAHA treatment or saline treatment andwere observed through immunostaining. Average optical signals forAc-lysine were measured on pyramidal neuron layer; SVP signals weremeasured from s.r. c. Images of Golgi staining from CA1 region ofhippocampus. For WT, naive, n=23; WT, SAHA, n=41; HDAC2OE, naïve, n=21;HDAC2OE, SAHA, n=32. Scale bar 10 μm. d. Memory test for mice withcontextual fear conditioning training (foot shock 0.5 mA) after 10 daySAHA injection (25 mg/kg, i.p.). WT mice (SAHA, n=10; saline, n=10) andHDAC2 KO mice (SAHA, n=8; saline, n=8). e. CA1 region from HDAC2KO micereceived chronic SAHA treatment or saline treatment and were observedthrough immunostaining. SVP-singles were quantified in the s.r. Saline,n=15; SAHA, n=22. Scale bar=50 μm. f. Images of Golgi staining of CA1region of hippocampus from HDAC2KO mice. HDAC2KO, SAHA, n=24; HDAC2KO,naïve, n=27. *, p<0.05; **, p<0.005; ***,p<0.001, unpaired studentt-test error bars indicate s.e.m.

FIG. 2 Increased α-Tubulin(K40) acetylation resulting from HDAC6inhibition does not facilitate associative learning in mice. a. Thestructure of WT-161 is shown. b. Selectivity of WT-161 (2 μM) forincreasing acetylated α-tubulin(K40) over total acetylated lysine(Ac-lysine) was measured in human MM1.S cells treated for 16 hrs andassessed for hyperacetylated histones and/or α-tubulin(K40) usingquantitative immunofluorescence imaging. Data presented are derived froma primary screen of a library of compounds biased for deacetylasefunction. c. Immunostaining of acetylated α-tubulin(K40) in area CA1 ofhippocampus from mice treated with WT-161 or SAHA (both conditions in 25mg/kg, i.p., 10 days) or saline is shown. Acetylated α-tubulin(K40)immunoreactive intensity signals in area CA1 were quantified (n=9, foreach group). **, p<0.005. d. Memory test of WT mice injected with SAHA(25 mg/kg) or WT-161 (25 mg/kg) for 10 days. Mice were subjected tocontextual fear conditioning training 24 hours before test (WT, n=20;SAHA, n=20; WT-161, n=10; ***, p<0.0005, student t-test).

FIG. 3 Expression and distribution of HDAC1 and HDAC2 in HDAC1OE andHDAC2OE mouse brain. a. Representative immunostaining images showing theexpression of HDAC1 in the WT and HDAC1OE mice brain are provided. In WTbrain, HDAC1 expression level is relatively higher in dentate gyrus thanother areas of the brain. Increased HDAC1 signal in HDAC1OE brain isdetected not only in the hippocampus but also in the cortex, amygdala(indicated with dashed lines) and basal forebrain. b. Representativeimmunostaining images showing the expression of HDAC2 in WT and HDAC2OEmice brain are presented. Scale bar, 400 μm. Scale bar for insertion,100 μm.

FIG. 4 HDAC2KO mice exhibit enhanced memory in behavior tasks. a. Escapelatency of WT, HDAC1OE and HDAC2OE mice in the visible platform watermaze test. Mice were trained in the swimming pool with a visibleplatform for 3 days, with two trials per training day. The latency formice to reach the platform was quantified (n=8 for each group). Allthree groups of mice reached the platform with similar escape latencieson the first day. No significant difference in escape latency wasdetected between the three groups of mice during the 3 days of training.b. Swimming speed in the water maze pool (n=8 for each group) is shown.c-d. Short-term memory was tested for WT, HDAC1OE and HDAC2OE mice incontextual- and tone-dependent fear conditioning paradigms (WT, n=9;HDAC2OE, n=9; HDAC1OE, n=8). No significant difference was detectedbetween the WT group and the HDAC1/2OE mice. e-f. Short-term memory wastested for HDAC2KO mice in contextual- and tone-dependent fearconditioning paradigms (WT, n=8; HDAC2KO, n=9). HDAC2KO mice showedsignificantly increased freezing in contextual fear conditioning(p=0.0100, compared to WT littermates), but not in tone-dependent fearconditioning (p=0.1439). g. Mean percent correct responses for WT (n=8)and HDAC2KO mice (n=10) during spatial non-matching to place testing onthe elevated T-maze is shown. HDAC2KO mice showed significant higheraccuracy during the training period (Block 2, p=0.044, Block 3,p=0.0087, student t-test; between genotypes, p=0.0252, two-way ANOVA).h. Mean percent correct responses for WT (n=8), HDAC1OE (n=7) andHDAC2OE (n=9) mice during spatial non-matching to place testing on theelevated T-maze is shown. HDAC2OE mice showed significant defects inaccuracy during training trail block 2 (p=0.0452, student t-test).

FIG. 5 Characterization of HDAC2KO mice. a. Schematic representation ofthe murine Hdac2 genomic locus is shown. Gray filled boxes indicateexons. Black arrowheads indicate loxP positions. P14F, P15R and P2 areoligo DNA primers used for genotyping. b. Westernblot analysis ofprotein lysates obtained from wild-type, Hdac2^(L/+) and Hdac2^(L/L)MEFs infected with either vector (V) or Cre-recombinase expressingretroviruses, using HDAC2 specific antibodies was performed. Cdk4 servedas a loading control. c. Observed and expected numbers and frequenciesof wild-type, Hdac2^(+/−) and Hdac2^(−/−) mice obtained from multipleHdac2^(+/−) intercrosses. d. Western blot analysis of HDAC1 and HDAC2expression levels in the brain lysate from the Hdac2^(−/−) mouse and WTlittermate was performed. HDAC1 expression level was increased inHdac2^(−/−) mice.

FIG. 6 SAHA treatment facilitates LTP in WT but not HDAC2KO hippocampus.a-b. One-month-old HDAC2KO mice and their WT littermates were injectedwith SAHA (25 mg/kg, i.p.) or saline for 10 days. An additionalinjection was introduced 30 minutes before sacrifice. Long-termpotentiation (LTP) was induced by one HFS stimulation (1×100 Hz, 1 s) ofSchaffer collaterals. a. A significant increase in the magnitude of LTPwas observed in the SAHA treated WT mice when compared to the salinegroup. b. No significant difference in the magnitude of LTP was detectedbetween SAHA and saline treated HDAC2KO mice. (**, p<0.005, two-wayANOVA).

FIG. 7 is a bar graph depicting the results of in vitro assays testingthe protective effects of HDAC over expression on p25 induced toxicity.Neurons were dissociated from E15.5 cortex and hippocampus andtransfected with plasmids encoding p25-GFP and Flag-HDACs at DIV4. 24hrs after transfection, neurons were fixed and processed for IHC. Allp25 positive neurons were counted, assuming most neurons are transfectedby both p25 and HDACs.

FIG. 8 is a table which shows the enzymatic inhibitory activity ofmultiple HDAC inhibitors against several of the known HDAC isoforms.

FIG. 9 shows the effects of HDAC inhibitors on histone acetylation marksin HeLa cell lysate. Series of compounds incubated with whole HEK293cells at 10 uM for a 6 hour time period. Western blot showing increasedacetylation levels over DMSO controls using anti-acetyl H4K12 antibodiesand horseradish peroxidase conjugated secondary antibody along with aluminol-based substrate. This demonstrates cellular HDAC activity ofthese analogs and the increase in acetylation in the specific mark,H4K12.

FIG. 10 is the quantification of the raw western data shown in FIG. 9.Relative to the DMSO control, multiple selectivity profiles areeffective in increasing H4K12 acetylation levels. This demonstrates thatHDAC 1,2 and HDAC 1,2,3 selective inhibitors have robust HDAC activityin whole cells on a specific histone loci (H4K12). BRD-9853 showsminimal activity in this cell line. BRD-4097 is the negative control.This is a benzamide with minimal HDAC inhibitory activity.

FIG. 11 is the quantification of the raw western blots used to measurethe effects of HDAC inhibitors on histone acetylation marks in HeLa celllysate. Relative to the DMSO control, there are varying degrees ofacetylation. The histogram demonstrates that HDAC1,2 and HDAC1,2,3selective compounds are effective at increasing the acetylation at theH4K12 loci.

FIG. 12 shows the increased H4K12 acetylation in mouse primary striatalcells. A. Western blots of primary striatal cells isolated from mousebrain that have been treated with HDAC inhibitors. Two sets of data with3 independent samples/set. B. Histograms represent the quantification ofwesterns shown in panel A.

FIG. 13 shows that treatment of neuronal cells with BRD-6929 andBRD-5298 enhances H4 and H2B histone acetylation in vitro.

FIG. 14 demonstrates the nuclear intensity of increasedH4K12-acetylation in mouse primary neuronal cultures. A. Controldemonstrating that BRD-6929 at 1 and 10 uM does not cause an increase ordecrease in overall cell number after 6 h incubation in brain regionspecific primary cultures (cortex and striatum). B. Histograms showingthat BRD-6929 at 10 uM causes an increase in H4K12 acetylation after 6 hincubation in to brain region specific primary cultures (striatum).Thus, An HDAC 1,2 selective compound is effective at increasingacetylation at a specific histone locus (H4K12) in cultured striatalneurons.

FIG. 15 demonstrates that an HDAC 1,2 selective compound cansignificantly increase acetylation marks associated with memory andlearning in neuronal cells isolated from specific brain regions andanalyzed using immunofluorescence. A. Control demonstrating thatBRD-6929 at 1 and 10 uM does not cause an increase or decrease inoverall cell number after 6 h incubation in brain region specificprimary cultures (striatum). B. Histograms showing that BRD-6929 at 1and 10 uM causes a 2-3 fold increase in H2B tetra-acetylation after 6 hincubation in brain region specific primary cultures (striatum). Thiseffect is significant relative to the DMSO control in all instances.

FIG. 16 demonstrates that HDAC 1,2 selective compounds are effective inincreasing the acetylation at the specific histone locus H2B. A.Micrograph showing the increased fluorescence in primary neuronal cellsafter treatment with DMSO or 10 uM BRD-5298, an HDAC 1,2 selectiveinhibitor, after 6 h incubation. The increased magenta fluorescencecorresponds to increased levels of H2B acetylation. B. Controldemonstrating that BRD-6929 and BRD-5298 at 1 and 10 uM do not cause anincrease or decrease in overall cell number after 6 h incubation inprimary neuronal cell cultures. C. Histograms showing that BRD-6929 andBRD-5298 (HDAC1,2 selective inhibitors) at 1 and 10 uM cause asignificant increase in H2B acetylation after 6 h incubation in primaryneuronal cell cultures.

FIG. 17 is the concentration-time curve of BRD-6929 in plasma and brainfollowing single 45 mg/kg i.p. dose in mice.

FIG. 18 is the experimental protocol for acute treatment with BRD-6929and the corresponding effects on histone acytlation in brain specificregions of adult male C57BL/6J mice.

FIG. 19 shows that acute treatment with BRD-6929 causes H2B (tetra)histone acetylation in cortex of adult male C57BL/6J mice. Thehistograms on the left are the quantification of the western gel datashown on the right. The data has been normalized to the level of histoneH3 levels. BRD-6929 causes a 1.5-2 fold increase in cortex for thismark. This demonstrates that BRD-6929 is a functional inhibitor of HDACsin the cortex after a single dose given systemically.

FIG. 20 shows that acute treatment with BRD-6929 causes increased H2BK5histone acetylation in cortex of adult male C57BL/6J mice. In cortexafter 1 hour, BRD-6929 causes a 1.5-2 fold increase in the acetylationlevels for H2BK5. This acetylation mark has been associated withincreased learning and memory.

FIG. 21 demonstrates the increase in acetylation marks in whole brainafter chronic administration of BRD-6929.

FIG. 22 demonstrates that BD-6929 increased associative learning andmemory in WT C57/BL6 mice.

DETAILED DESCRIPTION

Increased histone-tail acetylation induced by inhibitors of histonedeacetylases (HDACis) facilitates learning and memory in wildtype mice,as well as in mouse models of neurodegeneration. Harnessing thetherapeutic potential of HDACis requires knowledge of the specific HDACfamily members linked to cognitive enhancement. It is shown according toaspects of the invention that neuron-specific overexpression of HDAC2,but not HDAC1, reduced dendritic spine density, synapse number, synapticplasticity, and memory formation. Conversely, HDAC2 deficiency resultedin increased synapse number and memory facilitation, similar to chronicHDAC inhibitor treatment in mice. Notably, reduced synapse number andlearning impairment of HDAC2 overexpressing mice was completelyameliorated by chronic HDACi treatment. Correspondingly, HDACi treatmentfailed to further facilitate memory formation in HDAC2 deficient mice.Furthermore, analysis of promoter occupancy revealed HDAC2 associateswith the promoter of genes implicated in synaptic plasticity and memoryformation. Our results suggest that HDAC2 plays a role in modulatinglong lasting changes of the synapse, which in turn negatively regulateslearning and memory.

The invention relates in some aspects to therapeutics for enhancingand/or retrieving memories as well as promoting learning and memory. A“memory” as used herein refers to the ability to recover informationabout past events or knowledge. Memories include short-term memory (alsoreferred to as working or recent memory) and long-term memory.Short-term memories involve recent events, while long-term memoriesrelate to the recall of events of the more distant past. Enhancing orretrieving to memories is distinct from learning. However, in someinstances in the art learning is referred to as memory. The presentinvention distinguishes between learning and memory and is focused onenhancing memories. Learning, unlike memory enhancement, refers to theability to create new memories that had not previously existed. In someinstances the invention also relates to methods for enhancing learning.Thus in order to test the ability of a therapeutic agent to effect theability of a subject to learn rather than recall old memories, thetherapeutic would be administered prior to or at the same time as thememory is created. In order to test the ability of a therapeutic toeffect recall of a previously created memory the therapeutic isadministered after the memory is created and preferably after the memoryis lost.

In some instances the invention relates to methods for recapturing amemory in a subject. In order to recapture the memory the memory hasbeen lost. A lost memory is one which cannot be retrieved by the subjectwithout assistance, such as the therapeutic of the invention. In otherwords the subject cannot recall the memory. As used herein the term“recapture” refers to the ability of a subject to recall a memory thatthe subject was previously unable to recall. Generally, such a subjecthas a condition referred to as memory loss. A subject having memory lossis a subject that cannot recall one or more memories. The memories maybe short term memories or long term memories. Methods for assessing theability to recall a memory are known to those of skill in the art andmay include routine cognitive tests.

In other instances the invention relates to a method for accessinglong-term memory in a subject having diminished access to a long-termmemory. A subject having diminished access to a memory is a subject thathas experienced one or more long term memory lapses. The long-termmemory lapse may be intermittent or continuous. Thus, a subject havingdiminished access to a long term memory includes but is not limited to asubject having memory loss, with respect to long term memories.

In some instances the long-term memory of the “subject having diminishedaccess” may be impaired. An impaired long-term memory is one in which aphysiological condition of the subject is associated with the long-termmemory loss. Conditions associated with long-term memory loss includebut are not limited to age related memory loss and injury related memoryloss.

As used herein “age related memory loss” refers to refers to any of acontinuum of conditions characterized by a deterioration of neurologicalfunctioning that does not rise to the level of a dementia, as furtherdefined herein and/or as defined by the Diagnostic and StatisticalManual of Mental Disorders: 4th Edition of the American PsychiatricAssociation (DSM-IV, 1994). This term specifically excludes age-relateddementias such as Alzheimer's disease and Parkinson's disease, andconditions of mental retardation such as Down's syndrome. Age relatedmemory loss is characterized by objective loss of memory in an oldersubject compared to his or her younger years, but cognitive testperformance that is within normal limits for the subject's age. Agerelated memory loss subjects score within a normal range on standardizeddiagnostic tests for dementias, as set forth by the DSM-IV. Moreover,the DSM-IV provides separate diagnostic criteria for a condition termedAge-Related Cognitive Decline. In the context of the present invention,as well as the terms “Age-Associated Memory Impairment” and“Age-Consistent Memory Decline” are understood to be synonymous with theage related memory loss. Age-related memory loss may include decreasedbrain weight, gyral atrophy, ventricular dilation, and selective loss ofneurons within different brain regions. For purposes of some embodimentsof the present invention, more progressive forms of memory loss are alsoincluded under the definition of age-related memory disorder. Thuspersons having greater than age-normal memory loss and cognitiveimpairment, yet scoring below the diagnostic threshold for frankdementia, may be referred to as having a mild neurocognitive disorder,mild cognitive impairment, late-life forgetfulness, benign senescentforgetfulness, incipient dementia, provisional dementia, and the like.Such subjects may be slightly more susceptible to developing frankdementia in later life (See also US patent application 2006/008517,which is incorporated by reference). Symptoms associated withage-related memory loss include but are not limited to alterations inbiochemical markers associated with the aging brain, such as IL-1beta,IFN-gamma, p-JNK, p-ERK, reduction in synaptic activity or function,such as synaptic plasticity, evidenced by reduction in long termpotentiation, diminution of memory and reduction of cognition.

As used herein “injury related memory loss” refers to damage whichoccurs to the brain, and which may result in neurological damage.Sources of brain injury include traumatic brain injury such asconcussive injuries or penetrating head wounds, brain tumors,alcoholism, Alzheimer's disease, stroke, heart attack and otherconditions that deprive the brain of oxygen, meningitis, AIDS, viralencephalitis, and hydrocephalus.

A subject shall mean a human or vertebrate animal or mammal includingbut not limited to a dog, cat, horse, cow, pig, sheep, goat, turkey,chicken, and primate, e.g., monkey. Subjects are those which are nototherwise in need of an HDAC inhibitor. Subjects specifically excludesubjects having Alzheimer's disease, except in the instance where asubject having Alzheimer's disease is explicitly recited.

The methods of the invention generally relate to methods for enhancingmemories. Methods for enhancing memories include reestablishing accessto memories as well as recapturing memories. The term re-establishingaccess as used herein refers to increasing retrieval of a memory.Although Applicants are not bound by a mechanism of action, it isbelieved that the compounds of the invention are effective in increasingretrieval of memories by re-establishing a synaptic network. The processof re-establishing a synaptic network may include an increase in thenumber of active brain synapses and or a reversal of neuronal loss.

As used herein, the term re-establish access to long-term memory whenused with respect to a disorder comprising memory loss or memory lapserefers to a treatment which increases the ability of a subject to recalla memory. In some instances the therapeutic of the invention alsodecreases the incidence and/or frequency with which the memory is lostor cannot be retrieved.

A subject in need of enhanced memories is one having memory loss ormemory lapse. The memory loss may occur by any mechanism, such as it maybe age related or caused by injury or disorders associated withcognitive impairment. Disorders associated with cognitive impairmentinclude for instance MC1 (mild cognitive impairment), Alzheimer'sDisease, memory loss, attention deficit symptoms associated withAlzheimer disease, neurodegeneration associated with Alzheimer disease,dementia of mixed vascular origin, dementia of degenerative origin,pre-senile dementia, senile dementia, dementia associated withParkinson's disease, vascular dementia, progressive supranuclear palsyor cortical basal degeneration.

Alzheimer's disease is a degenerative brain disorder characterized bycognitive and noncognitive neuropsychiatric symptoms, which accounts forapproximately 60% of all cases of dementia for patients over 65 yearsold. In Alzheimer's disease the cognitive systems that control memoryhave been damaged. Often long-term memory is retained while short-termmemory is lost; conversely, memories may become confused, resulting inmistakes in recognizing people or places that should be familiar.Psychiatric symptoms are common in Alzheimer's disease, with psychosis(hallucinations and delusions) present in many patients. It is possiblethat the psychotic symptoms of Alzheimer's disease involve a shift inthe concentration of dopamine or acetylcholine, which may augment adopaminergic/cholinergic balance, thereby resulting in psychoticbehavior. For example, it has been proposed that an increased dopaminerelease may be responsible for the positive symptoms of schizophrenia.This may result in a positive disruption of the dopaminergic/cholinergicbalance. In Alzheimer's disease, the reduction in cholinergic neuronseffectively reduces acetylcholine release resulting in a negativedisruption of the dopaminergic/cholinergic balance. Indeed,antipsychotic agents that are used to relieve psychosis of schizophreniaare also useful in alleviating psychosis in Alzheimer's patients andcould be combined with the compositions described herein for use in themethods of the invention.

Methods for recapturing a memory in a subject having Alzheimer's diseaseby administering an HDAC inhibitor are also provided according to theinvention. Such methods optionally administering the inhibitor andmonitoring the subject to identify recapture of a memory that waspreviously lost. Subjects may be monitored by routine tests known in theart. For instance some are described in books such as DSM describedabove or in the medical literature.

Vascular dementia, also referred to as “multi-infarct dementia”, refersto a group of syndromes caused by different mechanisms all resulting invascular lesions in the brain. The main subtypes of vascular dementiaare, for example vascular mild cognitive impairment, multi-infarctdementia, vascular dementia due to a strategic single infarct (affectingthe thalamus, the anterior cerebral artery, the parietal lobes or thecingulate gyrus), vascular dementia due to hemorrhagic lesions, smallvessel disease (including, e.g. vascular dementia due to lacunar lesionsand Binswanger disease), and mixed Alzheimer's Disease with vasculardementia.

HDACs interact with other chromatin-modifying enzymes and co-regulatorsand play a key role in shaping epigenetic landscapes (Goldberg, A. D.,Allis, C. D., & Bernstein, E. Cell 128 (4), 635-638 (2007).). There area total of 18 HDAC enzymes in the mammalian genome, which are generallydivided into four classes including class I, II, III and IV. Theseenzymes are known to have both histone and non-histone substrates. Withthe exception of the class II HDAC5, which has recently been implicatedin the response to both antidepressant action (Tsankova, N. M. et al.Nat Neurosci 9 (4), 519-525 (2006).) and chronic emotional stimuli(Renthal, W. et al. Neuron 56 (3), 517-529 (2007).), little is knownabout the function of HDACs in the brain. Among the HDACs, Class I, IIand IV HDACs are the zinc-dependent hydrolases. Class I HDACs include 1,2, 3, and 8, which have been well documented to exert deacetylaseactivity on histone substrates as well as non-histone substrates. Thesefamily members are all inhibited by the non-selective HDAC inhibitorsodium butyrate. Class II HDACs can be divided into Class IIa members,which include HDAC 4, 5, 7 and 9, and Class IIb members, which includeHDAC6 and 10. In the case of HDAC5, a role in the brain has beenidentified in response to both antidepressant action and to chronicemotional stimuli. However, whether class IIa HDACs themselves havefunctional histone (or other non-histone) deacetylates activity, ratherthan activity contributed by co-purifying class I HDACs, currentlyremains unclear. Class IIb family members, HDAC6 and 10 are mainlylocalized in the cytoplasm. HDAC6 is unique in the family in itspossession of two deacetylase domains. HDAC6 has been shown to functionas both an α-tublin (K40) deacetylase and to regulateubiquitin-dependent protein degradation by the proteasome. In contrast,class III HDACs (sirtuins; SIRT1-7) are non-classical, NAD(+)-dependentenzymes, which exhibit a non-overlapping sensitivity to most structuralclasses of inhibitors of zinc-dependent HDACs, including SB. The latterfinding suggests the sirtuins are not the relevant targets of HDACiinduced memory enhancement.

The compounds useful according to the invention are HDAC2 inhibitors. AnHDAC2 inhibitor as used herein is any compound, including proteins,small molecules, and nucleic acids, that reduces HDAC2 activity and/orexpression. HDAC2 inhibitors may in some embodiments be selective HDAC2inhibitors. A selective HDAC2 inhibitor is a compound that inhibits theactivity or expression of HDAC2 but does not significantly inhibit theactivity or expression of at least 2 other HDAC enzymes such as HDAC1,HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, HDAC12,HDAC13, HDAC14, HDAC15, HDAC16, HDAC17, or HDAC18. In some embodiments aselective HDAC2 inhibitor is a compound that inhibits the activity orexpression of HDAC2 but does not significantly inhibit the activity orexpression of any other HDAC enzymes. In other embodiments a selectiveHDAC2 inhibitor does not significantly inhibit the activity orexpression of any other class I HDAC enzymes. An HDAC1/HDAC2 selectiveinhibitor is a compound that inhibits the activity or expression ofHDAC1 and HDAC2 but does not significantly inhibit the activity orexpression of at least one non-class I HDAC enzyme. In some embodimentsan HDAC1/HDAC2 selective inhibitor does not significantly inhibit theactivity or expression of any non-class I HDAC enzyme. In otherembodiments an HDAC1/HDAC2 selective inhibitor does not significantlyinhibit the activity or expression of a HDAC3 enzyme. AnHDAC1/HDAC2/HDAC3 selective inhibitor is a compound that inhibits theactivity or expression of HDAC1 and HDAC2 and HDAC3 but does notsignificantly inhibit the activity or expression of at least onenon-class I HDAC enzyme. In some embodiments an HDAC1/HDAC2/HDAC3selective inhibitor does not significantly inhibit the activity orexpression of any non-class I HDAC enzyme. Significantly inhibit refersto an amount that would detectably alter the activity of the HDAC in acell such as in vivo. In some embodiments the non-selective HDAC2inhibitor may be partially selective. For instance, it may act as aninhibitor of one or more other enzymes of HDAC1-HDAC18 but not all.Preferably the HDAC inhibitor does not act as an inhibitor of HDAC1,HDAC5, HDAC6, HDAC7, and HDAC10. In some embodiments, the HDAC2inhibitor is a selective HDAC1/HDAC2/HDAC10 inhibitor. In someembodiments, the selective HDAC1/HDAC2/HDAC10 inhibitor is BRD-6929. Inother embodiments, the HDAC2 inhibitor is a selectiveHDAC1/HDAC2/HDAC3/HDAC10 inhibitor.

HDAC2 inhibitors include binding peptides such as antibodies, preferablymonoclonal antibodies, antibody fragments, scFv, etc that specificallyreact with the histone deacetylase, small molecule inhibitors (oftenclassically referred to as HDAC inhibitors), and expression inhibitorssuch as antisense and siRNA.

Studies described in the Examples below were also undertaken todetermine which of the 11 histone deacetylases is responsible for theobserved function and to identify selective HDAC inhibitors forenhancing memory. It had been discovered that while HDAC1 Tg mice do notshow any difference in learning behavior compared to the control mice,HDAC2 Tg mice have impaired learning as evaluated by Pavlovian fearconditioning and Morris water maze tests. Remarkably, HDAC2 neuronspecific knockout mice (loss of function) display enhanced learning.Conversely, MS-275, a class 1 HDAC inhibitor (HDAC1/HDAC3 specific), didnot facilitate associative learning in mice and MS-275 treated miceshowed lower number of c-fos positive cells after fear conditioningtraining compared to saline treated group. Additional data alsodemonstrates that HDAC5, HDAC6, HDAC7 and HDAC10 are not useful forenhancing memory. These observations suggest that HDAC2 participates inlearning and memory and that it is likely to be the target of inhibitionby the general HDAC inhibitors. Even more surprisingly it was discoveredthat HDAC1/HDAC2 and HDAC1/HDAC2/HDAC3 selective inhibitors were alsouseful in enhancing learning and memory. Prior studies by some of theinstant inventors had demonstrated that HDAC1 activators promoteneurogenesis. Thus it was unexpected that HDAC1/HDAC2 inhibitors wouldbe useful for enhancing memory.

HDAC inhibitors include but are not limited to the following compounds,functional analogs and salts thereof: trichostatin A (TSA), trichostatinB, trichostatin C, trapoxin A, trapoxin B, chlamydocin, sodium salts ofbutyrate, butyric acid, sodium salts of phenylbutyrate, phenylbutyricacid, scriptaid, FR901228, depudecin, oxamflatin, pyroxamide, apicidinB, apicidin C, Helminthsporium carbonum toxin,2-amino-8-oxo-9,10-epoxy-decanoyl,3-(4-aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide, suberoylanilidehydroxamic acid (SAHA), valproic acid, FK228, or m-carboxycinnamic acidbis-hydroxamide. In preferred embodiments the HDAC inhibitor is an HDAC2inhibitor such as sodium butyrate, SAHA or TSA. Derivatives of theinhibitors showing increased pharmacological half-life are also usefulaccording to the invention (Brettman and Chaturvedi, J. Cli. Pharmacol.36 (1996), 617-622).

An example of a pan or universal HDAC inhibitor is SAHA. “SAHA” as usedherein refers to suberoylanilide hydroxamic acid, analogs, derivativesand polymorphs. Polymorphs of SAHA are described in US Published PatentApplication No. 20040122101 which is incorporated by reference.

HDAC2 inhibitors, including HDAC2 selective inhibitors, HDAC1/HDAC2selective inhibitors and HDAC1/HDAC2/HDAC3 selective inhibitors, of theinvention include small molecules as well as inhibitory nucleic acidssuch as antisense and siRNA. Small molecule HDAC2 inhibitors include forinstance compounds of the following formulas:

wherein R₁ and R₂ are independently selected from H, substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₁₋₆alkyl,heterocyclyl, C₁₋₆alkylene, heteroaryl, heteroarylene, andheteroarylene-alkylene; and R₃ is aryl or heteroaryl. In someembodiments R₁ is unsubstituted acyclic C₁₋₆alkyl; R₁ is selected from agroup consisting of methyl, ethyl, propyl, and butyl; R₁ isheteroarylene-alkylene; R₁ is heteroarylene-C₁₋₆alkylene; R₁ ispyridinyl-ethylene; R₂ is hydrogen; R₃ is heteroaryl; or R₃ is thienyl.

wherein R₁ and R₂ are independently selected from H, substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₁₋₆alkyl,heterocyclyl, C₁₋₆alkylene, heteroaryl, heteroarylene,heteroarylene-alkylene, arylene-alkylene; and heterocyclyl-alkyleneoptionally substituted; and R₃ is aryl or heteroaryl. In someembodiments R₁ is unsubstituted acyclic C₁₋₆alkyl; R₁ is selected from agroup consisting of methyl, ethyl, propyl, and butyl; R₁ isheteroarylene-alkylene; R₁ is heteroarylene-C₁₋₆alkylene; R₁ ispyridinyl-ethylene; R₁ is arylene-alkylene; R₁ is arylene-C₁₋₆alkylene;R₁ is phenyl-ethylene; R₁ is heterocyclyl-alkylene; R₁ is unsubstitutedheterocyclyl-C₁₋₆alkylene; R₁ is piperazine-ethylene; R₁ is substitutedheterocyclyl-C₁₋₆alkylene; R₁ is substituted piperazine-ethylene; R₁ isC₁₋₆alkylene substituted piperazine-ethylene; R₁ is methyl substitutedpiperazine-ethylene; R₂ is hydrogen; R₃ is heteroaryl; R₃ is thienyl; orR₃ is pyridinyl.

wherein X is —C(O)—N(R₁)(R₂),C₁₋₆alkylene-N(H)—C₁₋₆alkylene-N(R₁)C(O)(R₂); or —N(R₁)C(O)R₂; R₁ and R₂are independently selected from H, and substituted or unsubstituted,branched or unbranched, cyclic or acyclic C₁₋₆alkyl; and R₃ is alkynyl,aryl, or heteroaryl. In some embodiments X is —C(O)—N(R₁)(R₂); R₁ and R₂are independently selected from H, unsubstituted, unbranched, acyclicC₁₋₆alkyl; R₁ and R₂ are independently selected from H, methyl, ethyl,propyl, and butyl; R₁ is H; R₁ and R₂ are H; X is —C(O)—NH₂; X isC₁₋₆alkylene-N(R₁)—C₁₋₆alkylene-N(R₁)C(O)(R₂); R₁ is H; X isC₁₋₆alkylene-N(H)—C₁₋₆alkylene-N(H)C(O)(R₂); X is —N(R₁)C(O)R₂; R₁ is H;R₁ is unsubstituted acyclic C₁₋₆alkyl; R₁ is selected from a groupconsisting of methyl, ethyl, propyl, and butyl; R₂ is unsubstitutedacyclic C₁₋₆alky; R₂ is selected from a group consisting of methyl,ethyl, propyl, and butyl; R₃ is heteroaryl; R₃ is thienyl; R₃ is aryl;R₃ is alkynyl; R₃ is C₁₋₆alkynyl; or R₃ is ethynyl.

wherein R₁ and R₂ are independently selected from H, and—C(O)—C₁₋₆alkyl; R₃ is optionally substituted aryl, optionallysubstituted heteroaryl, or aryl-C₁₋₆alkylene. In some embodiments R₁ isH; R₁ and R₂ are H; R₁ is —C(O)—C₁₋₆alkyl; R₁ is —C(O)-methyl; R₁ is—C(O)-methyl and R₂ is H; R₃ is optionally substituted aryl; R₃ istolyl; R₃ is optionally substituted heteroaryl; R₃ is thienyl; R₃ isaryl-C₁₋₆alkylene; or R₃ is phenyl-ethylene.

wherein R₁ and R₂ are independently selected from H, and substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₁₋₆alkyl; andR₃ is aryl or heteroaryl. In some embodiments R₁ is H; R₁ and R₂ are H;R₁ is methyl, ethyl, propyl, to or butyl; R₃ is aryl; R₃ is heteroaryl;or R₃ is thienyl.

wherein R₁ and R₂ are independently selected from H, substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₁₋₆alkyl,heterocyclyl, heteroaryl, aryl, and aryl-C₁₋₆alkylene. In someembodiments R₁ is H; R₁ and R₂ are H: R₁ is methyl, ethyl, propyl, orbutyl; R₁ is aryl-C₁₋₆alkylene; R₁ is phenyl-ethylene; or R₂ is H.

“Alkyl” in general, refers to an aliphatic hydrocarbon group which maybe straight, branched or cyclic having from 1 to about 10 carbon atomsin the chain, and all combinations and sub combinations of rangestherein. The term “alkyl” includes both “unsubstituted alkyls” and“substituted alkyls,” the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thebackbone. In preferred embodiments, a straight chain or branched chainalkyl has 12 or fewer carbon atoms in its backbone (e.g., C₁-C₁₂ forstraight chain, C₃-C₁₂ for branched chain), and more preferably 6 orfewer, and even more preferably 4 or fewer. Likewise, preferredcycloalkyls have from 3-10 carbon atoms in their ring structure, andmore preferably have 5, 6 or 7 carbons in the ring structure. Unless thenumber of carbons is otherwise specified, “lower alkyl” as used hereinmeans an alkyl group, as defined above, but having from one to tencarbons, more preferably from one to six carbon atoms in its backbonestructure, and even more preferably from one to four carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl. Alkyl groups include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl,cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl, cyclohexyl,cyclooctyl, adamantyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. Alkyl substituents can include, for example, alkenyl,alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “alkenyl” refers to unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

As used herein, the term “halogen” designates —F, —Cl, —Br or —I; theterm “sulfhydryl” means —SH; and the term “hydroxyl” means —OH.

The term “aryl,” alone or in combination, means a carbocyclic aromaticsystem containing one, two or three rings wherein such rings may beattached together in a pendent manner or may be fused. The term “aryl”embraces aromatic radicals such as phenyl, naphthyl, tetrahydronapthyl,indane and biphenyl, and includes carbocyclic aryl, heterocyclic aryland biaryl groups, all of which may be optionally substituted. The term“aryl” as used herein includes 5-, 6- and 7-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, benzene, pyrrole, furan, thiophene, imidazole, oxazole,thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine andpyrimidine, and the like. Those aryl groups having heteroatoms in thering structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The term “biaryl” represents aryl groups which have 5-14 atomscontaining more than one aromatic ring including both fused ring systemsand aryl groups substituted with other aryl groups. Such groups may beoptionally substituted. Suitable biaryl groups include naphthyl andbiphenyl. The term “carbocyclic” refers to a cyclic compounds in whichall of the ring members are carbon atoms. Such rings may be optionallysubstituted. The compound can be a single ring or a biaryl ring. Theterm “cycloalkyl” embraces radicals having three to ten carbon atoms,such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyland norboryl. Such groups may be substituted.

“Heterocyclic” aryl or “heteroaryl” groups are groups which have 5-14ring atoms wherein 1 to 4 heteroatoms are ring atoms in the aromaticring and the remainder of the ring atoms being carbon atoms. Suitableheteroatoms include oxygen, sulfur, and nitrogen. Suitable heteroarylgroups include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkylpyrrolyl, pyridyl-N-oxide, pyrimidyl, pyrazinyl, imidazolyl, indolyl andthe like, all optionally substituted. The term “heterocyclic” refers tocyclic compounds having as ring members atoms of at least two differentelements. The compound can be a single ring or a biaryl. Heterocyclicgroups include, for example, thiophene, benzothiophene, thianthrene,furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole,imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring can be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

Non-limiting examples of HDAC2 inhibitors useful in the methods of theinvention are:

The compounds of the invention may optionally be administered with othercompounds such as DNA methylation inhibitors. A DNA methylationinhibitor is an agent that directly or indirectly causes a reduction inthe level of methylation of a nucleic acid molecule. DNA methylationinhibitors are well known and routinely utilized in the art and include,but are not limited to, inhibitors of methylating enzymes such asmethylases and methyltransferases. Non-limiting examples of DNAmethylation inhibitors include 5-azacytidine, 5-aza-2′deoxycytidine(also known as Decitabine in Europe), 5,6-dihydro-5-azacytidine,5,6-dihydro-5-aza-2′deoxycytidine, 5-fluorocytidine,5-fluoro-2′deoxycytidine, and short oligonucleotides containing5-aza-2′deoxycytosine, 5,6-dihydro-5-aza-2′deoxycytosine, and5-fluoro-2′deoxycytosine, and procainamide, Zebularine, and(−)-egallocatechin-3-gallate.

In addition to the classic small molecule HDAC inhibitors describedabove, HDAC2 can also be inhibited by nucleic acid based or expressioninhibitors such as antisense and RNAi. Thus, the invention embracesinhibitory nucleic acids such as antisense oligonucleotides thatselectively bind to nucleic acid molecules encoding HDAC2 to decreaseexpression and activity of this protein.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene or transcript. Antisenseoligonucleotides that selectively bind to a nucleic acid moleculeencoding a histone deacetylase are particularly preferred. Those skilledin the art will recognize that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence.

It is preferred that the antisense oligonucleotide be constructed andarranged so as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon the nucleotide sequences of nucleic acid tomolecules encoding histone deacetylase, (e.g., GenBank Accession NosNP_(—)848512, NP_(—)848510, NP_(—)478057, NP_(—)478056, NP_(—)055522) orupon allelic or homologous genomic and/or cDNA sequences, one of skillin the art can easily choose and synthesize any of a number ofappropriate antisense molecules for use in accordance with the presentinvention. In order to be sufficiently selective and potent forinhibition, such antisense oligonucleotides should comprise at leastabout 10 and, more preferably, at least about 15 consecutive bases whichare complementary to the target, although in certain cases modifiedoligonucleotides as short as 7 bases in length have been usedsuccessfully as antisense oligonucleotides. See Wagner et al., Nat. Med.1(11):1116-1118, 1995. Most preferably, the antisense oligonucleotidescomprise a complementary sequence of 20-30 bases. Althougholigonucleotides may be chosen which are antisense to any region of thegene or mRNA transcripts, in preferred embodiments the antisenseoligonucleotides correspond to N-terminal or 5′ upstream sites such astranslation initiation, transcription initiation or promoter sites. Inaddition, 3′-untranslated regions may be targeted by antisenseoligonucleotides. Targeting to mRNA splicing sites has also been used inthe art but may be less preferred if alternative mRNA splicing occurs.In addition, the antisense is targeted, preferably, to sites in whichmRNA secondary structure is not expected (see, e.g., Sainio et al., CellMol. Neurobiol. 14(5):439-457, 1994) and at which proteins are notexpected to bind.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acid molecules has beencovalently attached to the oligonucleotide. Preferred syntheticinternucleoside linkages are phosphorothioates, alkylphosphonates,phosphorodithioates, phosphate esters, alkylphosphonothioates,phosphoramidates, carbamates, carbonates, phosphate triesters,acetamidates, carboxymethyl esters and peptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose.

The present invention, thus, contemplates pharmaceutical preparationscontaining modified antisense molecules that are complementary to andhybridizable with, under physiological conditions, nucleic acidmolecules encoding a histone deacetylase, together with pharmaceuticallyacceptable carriers. Antisense oligonucleotides may be administered aspart of a pharmaceutical composition. In this latter embodiment, it maybe preferable that a slow intravenous administration be used. Such apharmaceutical composition may include the antisense oligonucleotides incombination with any standard physiologically and/or pharmaceuticallyacceptable carriers which are known in the art. The compositions shouldbe sterile and contain a therapeutically effective amount of theantisense oligonucleotides in a unit of weight or volume suitable foradministration to a subject.

The methods of the invention also encompass use of isolated short RNAthat directs the sequence-specific degradation of a histone deacetylasemRNA through a process known as RNA interference (RNAi). The process isknown to occur in a wide variety of organisms, including embryos ofmammals and other vertebrates. It has been demonstrated that dsRNA isprocessed to RNA segments 21-23 nucleotides (nt) in length, andfurthermore, that they mediate RNA interference in the absence of longerdsRNA. Thus, these 21-23 nt fragments are sequence-specific mediators ofRNA degradation and are referred to herein as siRNA or RNAi. Methods ofthe invention encompass the use of these fragments (or recombinantlyproduced or chemically synthesized oligonucleotides of the same orsimilar nature) to enable the targeting of histone deacetylase mRNAs fordegradation in mammalian cells useful in the therapeutic applicationsdiscussed herein.

The nucleotide sequence of HDAC2 is well known in the art and can beused by one of skill in the art using art recognized techniques incombination with the guidance set forth below to produce the appropriatesiRNA molecules. Such methods are described in more detail below.

In one embodiment, the invention features a siNA molecule having RNAiactivity against target HDAC2 RNA (e.g., coding or non-coding RNA),wherein the siNA molecule comprises a sequence complementary to anyHDAC2 RNA sequence, such as that sequence having HDAC2 GenBank AccessionNo: mRNA NM_(—)001527 for homo sapiens. Chemical modifications can beapplied to any siNA construct of the invention. As shown in GenBankAccession No: mRNA NM_(—)001527 the protein sequence of HDAC2 is:

(SEQ ID NO: 1) MRSPPCGLLRWFGGPLLASWCRCHLRFRAFGTSAGWYRAFPAPPPLLPPACPSPRDYRPHVSLSPFLSRPSRGGSSSSSSSRRRSPVAAVAGEPMAYSQGGGKKKVCYYYDGDIGNYYYGQGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPHKATAEEMTKYHSDEYIKFLRSIRPDNMSEYSKQMQRFNVGEDCPVFDGLFEFCQLSTGGSVAGAVKLNRQQTDMAVNWAGGLHHAKKSEASGFCYVNDIVLAILELLKYHQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYFPGTGDLRDIGAGKGKYYAVNFPMRDGIDDESYGQIFKPIISKVMEMYQPSAVVLQCGADSLSGDRLGCFNLTVKGHAKCVEVVKTFNLPLLMLGGGGYTIRNVARCWTYETAVALDCEIPNELPYNDYFEYFGPDFKLHISPSNMTNQNTPEYMEKIKQRLFENLRMLPHAPGVQMQAIPEDAVHEDSGDEDGEDPDKRISIRASDKRIACDEEFSDSEDEGEGGRRNVADHKKGAKKARIEEDKKETEDKKTDVKEEDKSKDNSGEKTDTKGTKSEQLSNP

The nucleic acid sequence is:

(SEQ ID NO: 2)   1 atgcgctcac ctccctgcgg cctcctgagg tggtttggtg gccccctcct cgcgagttgg  61 tgccgctgcc acctccgatt ccgagctttc ggcacctctg ccgggtggta ccgagccttc 121 ccggcgcccc ctcctctcct cccaccggcc tgccattccc cgcgggacta tcgcccccac 181 gtttccctca gcccttttct ctcccggccg agccgcggcg gcagcagcag cagcagcagc 241 agcaggagga ggagcccggt ggcggcggtg gccggggagc ccatggcgta cagtcaagga 301 ggcggcaaaa aaaaagtctg ctactactac gacggtgata ttggaaatta ttattatgga 361 cagggtcatc ccatgaagcc tcatagaatc cgcatgaccc ataacttgct gttaaattat 421 ggcttataca gaaaaatgga aatatatagg ccccataaag ccactgccga agaaatgaca 481 aaatatcaca gtgatgagta tatcaaattt ctacggtcaa taagaccaga taacatgtct 541 gagtatagta agcagatgca gagatttaat gttggagaag attgtccagt gtttgatgga 601 ctctttgagt tttgtcagct ctcaactggc ggttcagttg ctggagctgt gaagttaaac 661 cgacaacaga ctgatatggc tgttaattgg gctggaggat tacatcatgc taagaaatca 721 gaagcatcag gattctgtta cgttaatgat attgtgcttg ccatccttga attactaaag 781 tatcatcaga gagtcttata tattgatata gatattcatc atggtgatgg tgttgaagaa 841 gctttttata caacagatcg tgtaatgacg gtatcattcc ataaatatgg ggaatacttt 901 cctggcacag gagacttgag ggatattggt gctggaaaag gcaaatacta tgctgtcaat 961 tttccaatga gagatggtat agatgatgag tcatatgggc agatatttaa gcctattatc1021 tcaaaggtga tggagatgta tcaacctagt gctgtggtat tacagtgtgg tgcagactca1081 ttatctggtg atagactggg ttgtttcaat ctaacagtca aaggtcatgc taaatgtgta1141 gaagttgtaa aaacttttaa cttaccatta ctgatgcttg gaggaggtgg ctacacaatc1201 cgtaatgttg ctcgatgttg gacatatgag actgcagttg cccttgattg tgagattccc1261 aatgagttgc catataatga ttactttgag tattttggac cagacttcaa actgcatatt1321 agtccttcaa acatgacaaa ccagaacact ccagaatata tggaaaagat aaaacagcgt1381 ttgtttgaaa atttgcgcat gttacctcat gcacctggtg tccagatgca agctattcca1441 gaagatgctg ttcatgaaga cagtggagat gaagatggag aagatccaga caagagaatt1501 tctattcgag catcagacaa gcggatagct tgtgatgaag aattctcaga ttctgaggat1561 gaaggagaag gaggtcgaag aaatgtggct gatcataaga aaggagcaaa gaaagctaga1621 attgaagaag ataagaaaga aacagaggac aaaaaaacag acgttaagga agaagataaa1681 tccaaggaca acagtggtga aaaaacagat accaaaggaa ccaaatcaga acagctcagc1741 aacccctgaa tttgacagtc tcaccaattt cagaaaatca ttaaaaagaa aatattgaaa1801 ggaaaatgtt ttctttttga agacttctgg cttcatttta tactactttg gcatggactg1861 tatttatttt caaatggctt tttcgttttt gtttttcttg gcaagtttta ttgtgagttt1921 ttctaattat gaagcaaaat ttcttttctc caccatgctt tatgtgatag tatttaaaat1981 tgatgtgagt tattatgtca aaaaaactga tctattaaag aagtaattgg cctttctgag2041 ctgatttttc catcttttgt aattatcttt attaaaaaat tgtacttgga ttatcttttg2101 tctgtttatt actacaatat gaagtcttgt ttcagtggct aatgacatca tttctgtaga2161 cttacaatac actctaggtg aaagataatg attacagctt gaaagataac tatttgctgt2221 ttctttggga agagtattta tagtaattat tacttatctt tgcaatagaa attctaccac2281 cttgccctct atagcttagc cagttagtat cagtgaagat taacatccca ttacaattta2341 tgaaataata cagactctgc aattgagatg taggagttct ttgagttgac ccaaagattc2401 tcaaaattga aatggaaatt ctgaattgaa agaagaaact gaccagaact tgtattgacc2461 agacttgcct atagtatatt gctggtttaa aatggaacct gcagacaaaa cctgtttctt2521 ttactgcatt tacatggcat ccaggttcca ttattattta cgtgacatcc aggtttctaa2581 ctcaaggaaa taaacacaac tgatttatca ttcagcaact acttattgtg tgtctgccca2641 ttttaagact gtaggagtgt aactgaatat aatggaaaaa tgctttcact cagagcttac2701 actcagagct tacattctag tagcaggaag cagacaaatg ggggtaactc ctgctactaa2761 gatgtgcaaa gaagacaaaa cattttagga acttgccaaa atcagtgaaa tctccctttt2821 tgtcaggccc acattgattc ttttgagatt aaaaattaca gaatgccaga agataattca2881 gtcaaaagta tttctcttca gtgcagtaaa atattaaaag aaaaaatatt tctctacaag2941 cctcttaaat gtttcagaca ttcacaatag cacctagact tttgtaatga acagctgtgc3001 acccaccccg acttaacaaa tattttactt ttgctatatt tgcttcggtg tttttttctt3061 aaaatacacc gaaattgaag ctctctttgt gtactttgtc gtttgtaatc ctccctccct3121 cccagcctta agaagtaatc accattgtga ttattttatc catgtttttg tgaatatttt3181 acccatgttt ttgtactttt gctacatatt tgttatcaat ctgtaaacct ttatgacatt3241 aggaactaag aaacttagtc ccttcgttag ggggataatg aaatgtattt agtgtttgtg3301 aaacatagat ggtatgtatt tggacaattc tgtaactttg ctttttttat ttttattttt3361 ccatagctta ttggggaaca ggtggtgttt ggttacatga ttaagttctt tagtggtgat3421 ttgtgggatt ttggtggacc catcacccaa gcagtgtaca ctgcacccta tttgtaatct3481 tttatccctc gcccccctcc caccatgcct cccgtctacc atgatgatcc tgttttaaat3541 aagaaaatac catttcgcag gctccagatg ttctggcatc ctccctgtgg atttcccagt3601 gcctgcagct cacaggacaa caggggctgt ggtagagtca cctatgagat cctggagtag3661 tggatggagg agatggaaca gtgaagacgg aaactgagct cagtatccgg gtgccaggag3721 acaaaggccc tttgcttttt ttcatttaat attctgatct acccctgttg acacatgtta3781 agtatagttc attttgactg ctatgtatta tgttccattg tgtgaacata ctgaaattgt3841 acacttcaat actatactgg atctccttgg gtgtatttaa gaggttttgt ttttctaagt3901 agttggttat atacaactaa aacctcaaga gaactatcta aagcaatttc agcaaggtga3961 tttggtacag cattaataaa cagaaatcag taacacttag tgaccaagtc tgttggaaga4021 acaaagaccc ccatttgtaa taacaaaatt tttagaaata atatgtaaag aagctatggt4081 tcttgtgtct agtaaggtca atgtaacata gtaagatgtc agaataccct aatactttaa4141 aaaattcata taggataaaa atgatatttg aaattggcaa ggaaagacat tattttgtaa4201 gtggaattgg gacaacaact ggtaaccaaa tggaaaaccc agttttctgc cctccactag4261 aagatttaaa taggaaaaga taaaactacc aaaaacctaa actcttaaag caaatggatt4321 gaagaaggcc taagtgtgac accaaactca actataaaag atatatttga taacaaaaaa4381 aaaattagtt cagtggacca acaaaaactt agaagacaag tcaagaaaaa tgacaaaaga4441 cagagtggga ggcagatttg taactcatcc aggtcaaaag gctcatatct aaagatagta4501 gaggaacaaa atgtataagg atgtgaactg ggaaacaaat acatataaat agtttgtaaa4561 tatgaaaaga tctttaacct cagtaaataa aaagctatag agagacttat tttttaactt4621 agttttttaa acctattatg tttatttatt ttttcttttt ttgagacgga gtctcgctgt4681 tgcccaggct ggtgtgcaat ggcgcaatct cggctcactg caacctccgc ctcccaggtt4741 gaagccattc tcctgcctca gcctcctgag tagctgggat tacaggcgcc tgtcaccacg4801 cccagctaat tgttcgcatt tttagtagag acggggtttc actatgttgg ccaggctggc4861 ctcgaactcc tgacctcatg atccacccac cttggccttc caaagtgctg ggattacagg4921 tgtgagctgc cgcacctggc tgtttatctc ttttttttag agaaaggatc tcggtcaccc4981 aggatggagt gtggtagcct gatcatatct cactacatct tagaacttct aggcttaggg5041 tattctccca cctcagcctc ccaagtagct gggactacaa gtgtgcacca tcacacctag5101 ctgattttta catttttatt ttggagagat ggtgtctccc tgtgttgccc aggctggtca5161 caaactccta ggctcaagcg attctcctga ctcaggcatg agccaccgta cccggcctaa5221 acctatcatg ttacagactt agaaagcaac tattgtcaag tgtttgagga aactcaggtc5281 aggtttggta aactaagata ttaactcaag taaagctctt taattcattt aatgaaggtg5341 ccacattgtc tcagttctct atggcatggg tgaatgctgt tctaagtcag cattggtact5401 ctaagctagt tacatcatat ctaagctttg cccttctacc agagctgcta gcattctgtc5461 aatgggcaat tatttgaagt tcttacattg aagttagtca cctacatctt ctgtttttta5521 tggttttgat gtagtaatac tgctgaagtt tttttgataa ctgcgattca taatatttgt5581 tattcatatt gtgatataaa tgataagggc ttttgaaaac aagtagtaat catttcaata5641 gcttaggatc tccaccataa tcttaggaaa attactaacc tctgtgcctc agttgcttca5701 tcatttaaaa tgaggaaaat aatagtccct acttaatagg tttgttgtga ggattgagtt5761 aataacatag ttaatgctca gtaagggtta gctgctattt ttttttcttt tttttttgaa5821 agagtctcac tctgttgccc aggttggagt gcagtggcat gatcttggct cactgcatcc5881 tctgcttcct aggttcaggt gattctcatg cttcagcctc ccaagcagct gggagtacag5941 gtgtgcacca ccacacctgg ctaatttttg tatatttagt agagacgggt tctcaccata6001 ttgtccaggc tggtcttgaa ctccttacct caaatgatcc acccgcctgg gcctcccaaa6061 gtgctgggat tacaagcatg agccaccgcg cctggcttgc tattgttatg aggtaaaggt6121 agatagatgg gtgagagtgg tgccagggga agtgttaaat ttttgagtgt tcctttagat6181 gccagatggg ttgtatctga gccttttatt gcagtttgat gcctactagt gtgaagacta6241 ctaggtcata gtggatagag aagcaatctt ttggagacct gattttagca aggatacgaa6301 taatatttga caactttggg gggatcttga tgcctctgta atttactcaa ggataatctc6361 aagaaaaatg gcattaagta gattacagaa aaaatagaac tatcatattg ttattattgg6421 ctatttacat gagcaatgcg gagaaatgtt taggattaca gcatttagaa gcttctcaat6481 tgctgcattt cctcactgta ccacaagatg gcagatactg catttaaaat ttttttttct6541 gtgtgttttc tcttatagtc acttggtggc catgtaacaa gcagagcaac atgtattaac6601 agattctttt tgaatgcaat attggattaa aaactttgaa ttaaaaaaaa aaaaaaaaa

Thus the invention features the use of small nucleic acid molecules,referred to as small interfering nucleic acid (siNA) that include, forexample: microRNA (miRNA), small interfering RNA (siRNA),double-stranded RNA (dsRNA), and short hairpin RNA (shRNA) molecules. AnsiNA of the invention can be unmodified or chemically-modified. An siNAof the instant invention can be chemically synthesized, expressed from avector or enzymatically synthesized as discussed herein. The instantinvention also features various chemically-modified synthetic smallinterfering nucleic acid (siNA) molecules capable of modulating geneexpression or activity in cells by RNA interference (RNAi). The use ofchemically-modified siNA improves various properties of native siNAmolecules through, for example, increased resistance to nucleasedegradation in vivo and/or through improved cellular uptake.Furthermore, siNA having multiple chemical modifications may retain itsRNAi activity. The siNA molecules of the instant invention provideuseful reagents and methods for a variety of therapeutic applications.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) that prevent their degradation by serumribonucleases can increase their potency (see e.g., Eckstein et al.,International Publication No. WO 92/07065; Perrault et al, 1990 Nature344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren,1992, Trends in Biochem. Sci. 17, 334; Usman et al., InternationalPublication No. WO 93/15187; and Rossi et al., International PublicationNo. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; and Burgin et al.,supra; all of these describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules herein). Modifications which enhance their efficacy in cells,and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired. (All these publications are hereby incorporated by referenceherein).

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, T-H, nucleotide base modifications (for a review see Usmanand Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic AcidsSymp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugarmodification of nucleic acid molecules have been extensively describedin the art (see Eckstein et al., International Publication PCT No. WO92/07065; Perrault et al. Nature, 1990, 344, 565 568; Pieken et al.Science, 1991, 253, 314317; Usman and Cedergren, Trends in Biochem.Sci., 1992, 17, 334 339; Usman et al. International Publication PCT No.WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995,J. Biol. Chem., 270, 25702; Beigelman et al., International PCTpublication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824;Usman et al., molecule comprises one or more chemical modifications.

In one embodiment, one of the strands of the double-stranded siNAmolecule comprises a nucleotide sequence that is complementary to anucleotide sequence of a target RNA or a portion thereof, and the secondstrand of the double-stranded siNA molecule comprises a nucleotidesequence identical to the nucleotide sequence or a portion thereof ofthe targeted RNA. In another embodiment, one of the strands of thedouble-stranded siNA molecule comprises a nucleotide sequence that issubstantially complementary to a nucleotide sequence of a target RNA ora portion thereof, and the second strand of the double-stranded siNAmolecule comprises a nucleotide sequence substantially similar to thenucleotide sequence or a portion thereof of the target RNA. In anotherembodiment, each strand of the siNA molecule comprises about 19 to about23 nucleotides, and each strand comprises at least about 19 nucleotidesthat are complementary to the nucleotides of the other strand.

In some embodiments an siNA is an shRNA, shRNA-mir, or microRNA moleculeencoded by and expressed from a genomically integrated transgene or aplasmid-based expression vector. Thus, in some embodiments a moleculecapable of inhibiting mRNA expression, or microRNA activity, is atransgene or plasmid-based expression vector that encodes asmall-interfering nucleic acid. Such transgenes and expression vectorscan employ either polymerase II or polymerase III promoters to driveexpression of these shRNAs and result in functional siRNAs in cells. Theformer polymerase permits the use of classic protein expressionstrategies, including inducible and tissue-specific expression systems.In some embodiments, transgenes and expression vectors are controlled bytissue specific promoters. In other embodiments transgenes andexpression vectors are controlled by inducible promoters, such astetracycline inducible expression systems.

In some embodiments, a small interfering nucleic acid of the inventionis expressed in mammalian cells using a mammalian expression vector. Therecombinant mammalian expression vector may be capable of directingexpression of the nucleic acid preferentially in a particular cell type(e.g., tissue-specific regulatory elements are used to express thenucleic acid). Tissue specific regulatory elements are known in the art.Non-limiting examples of suitable tissue-specific promoters include themyosin heavy chain promoter, albumin promoter, lymphoid-specificpromoters, neuron specific promoters, pancreas specific promoters, andmammary gland specific promoters. Developmentally-regulated promotersare also encompassed, for example the murine hox promoters and theα-fetoprotein promoter.

Other inhibitor molecules that can be used include ribozymes, peptides,DNAzymes, peptide nucleic acids (PNAs), triple helix formingoligonucleotides, antibodies, and aptamers and modified form(s) thereofdirected to sequences in gene(s), RNA transcripts, or proteins.Antisense and ribozyme suppression strategies have led to the reversalof a tumor phenotype by reducing expression of a gene product or bycleaving a mutant transcript at the site of the mutation (Carter andLemoine Br. J. Cancer. 67(5):869-76, 1993; Lange et al., Leukemia.6(10:1786-94, 1993; Valera et al., J. Biol. Chem. 269(46):28543-6, 1994;Dosaka-Akita et al., Am. J. Clin. Pathol. 102(5):660-4, 1994; Feng etal., Cancer Res. 55(10):2024-8, 1995; Quattrone et al., Cancer Res.55(1):90-5, 1995; Lewin et al., Nat. Med. 4(8):967-71, 1998). Forexample, neoplastic reversion was obtained using a ribozyme targeted toan H-Ras mutation in bladder carcinoma cells (Feng et al., Cancer Res.55(10):2024-8, 1995). Ribozymes have also been proposed as a means ofboth inhibiting gene expression of a mutant gene and of correcting themutant by targeted trans-splicing (Sullenger and Cech Nature371(6498):619-22, 1994; Jones et al., Nat. Med. 2(6):643-8, 1996).Ribozyme activity may be augmented by the use of, for example,non-specific nucleic acid binding proteins or facilitatoroligonucleotides (Herschlag et al., Embo J. 13(12):2913-24, 1994; toJankowsky and Schwenzer Nucleic Acids Res. 24(3):423-9, 1996).Multitarget ribozymes (connected or shotgun) have been suggested as ameans of improving efficiency of ribozymes for gene suppression (Ohkawaet al., Nucleic Acids Symp Ser. (29):121-2, 1993).

Triple helix approaches have also been investigated forsequence-specific gene suppression. Triple helix formingoligonucleotides have been found in some cases to bind in asequence-specific manner (Postel et al., Proc. Natl. Acad. Sci. U.S.A.88(18):8227-31, 1991; Duval-Valentin et al., Proc. Natl. Acad. Sci.U.S.A. 89(2):504-8, 1992; Hardenbol and Van Dyke Proc. Natl. Acad. Sci.U.S.A. 93(7):2811-6, 1996; Porumb et al., Cancer Res. 56(3):515-22,1996). Similarly, peptide nucleic acids have been shown to inhibit geneexpression (Hanvey et al., Antisense Res. Dev. 1(4):307-17, 1991;Knudsen and Nielson Nucleic Acids Res. 24(3):494-500, 1996; Taylor etal., Arch. Surg. 132(11):1177-83, 1997). Minor-groove binding polyamidescan bind in a sequence-specific manner to DNA targets and hence mayrepresent useful small molecules for future suppression at the DNA level(Trauger et al., Chem. Biol. 3(5):369-77, 1996). In addition,suppression has been obtained by interference at the protein level usingdominant negative mutant peptides and antibodies (Herskowitz Nature329(6136):219-22, 1987; Rimsky et al., Nature 341(6241):453-6, 1989;Wright et al., Proc. Natl. Acad. Sci. U.S.A. 86(9):3199-203, 1989). Insome cases suppression strategies have led to a reduction in RNA levelswithout a concomitant reduction in proteins, whereas in others,reductions in RNA have been mirrored by reductions in protein.

The diverse array of suppression strategies that can be employedincludes the use of DNA and/or RNA aptamers that can be selected totarget, for example HDAC2. Suppression and replacement using aptamersfor suppression in conjunction with a modified replacement gene andencoded protein that is refractory or partially refractory toaptamer-based suppression could be used in the invention.

The methods for design of the RNA's that mediate RNAi and the methodsfor transfection of the RNAs into cells and animals is well known in theart and are readily commercially available (Verma N. K. et al, J. Clin.Pharm. Ther., 28(5):395-404(2004), Mello C. C. et al. Nature,431(7006)338-42 (2004), Dykxhoorn D. M. et al., Nat. Rev. Mol. Cell.Biol. 4(6):457-67 (2003) Proligo (Hamburg, Germany), Dharmacon Research(Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science,Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes(Ashland, Mass., USA), and Cruachem (Glasgow, UK)). The RNAs arepreferably chemically synthesized using appropriately protectedribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.Most conveniently, siRNAs are obtained from commercial RNA oligosynthesis suppliers listed herein. In general, RNAs are not toodifficult to synthesize and are readily provided in a quality suitablefor RNAi. A typical 0.2 μmol-scale RNA synthesis provides about 1milligram of RNA, which is sufficient for 1000 transfection experimentsusing a 24-well tissue culture plate format.

The histone deacetylase cDNA specific siRNA is designed preferably byselecting a sequence that is not within 50-100 bp of the start codon andthe termination codon, avoids intron regions, avoids stretches of 4 ormore bases such as AAAA, CCCC, avoids regions with GC content <30%or >60%, avoids repeats and low complex sequence, and it avoids singlenucleotide polymorphism sites. The histone deacetylase siRNA may bedesigned by a search for a 23-nt sequence motif AA(N19). If no suitablesequence is found, then a 23-nt sequence motif NA(N21) may be used withconversion of the 3′ end of the sense siRNA to TT. Alternatively, thehistone deacetylase siRNA can be designed by a search for NAR(N17)YNN.The target sequence may have a GC content of around 50%. The siRNAtargeted sequence may be further evaluated using a BLAST homology searchto avoid off target effects on other genes or sequences. Negativecontrols are designed by scrambling targeted siRNA sequences. Thecontrol RNA preferably has the same length and nucleotide composition asthe siRNA but has at least 4-5 bases mismatched to the siRNA. The RNAmolecules of the present invention can comprise a 3′ hydroxyl group. TheRNA molecules can be single-stranded or double stranded; such moleculescan be blunt ended or comprise overhanging ends (e.g., 5′, 3′) fromabout 1 to about 6 nucleotides in length (e.g., pyrimidine nucleotides,purine nucleotides). In order to further enhance the stability of theRNA of the present invention, the 3′ overhangs can be stabilized againstdegradation. The RNA can be stabilized by including purine nucleotides,such as adenosine or guanosine nucleotides. Alternatively, substitutionof pyrimidine nucleotides by modified analogues, e.g., substitution ofuridine 2 nucleotide 3′ overhangs by 2′-deoxythymidine is tolerated anddoes not affect the efficiency of RNAi. The absence of a 2′ hydroxylsignificantly enhances the nuclease resistance of the overhang in tissueculture medium.

The RNA molecules used in the methods of the present invention can beobtained using a number of techniques known to those of skill in theart. For example, the RNA can be chemically synthesized or recombinantlyproduced using methods known in the art. Such methods are described inU.S. Published Patent Application Nos. US2002-0086356A1 andUS2003-0206884A1 that are hereby incorporated by reference in theirentirety.

Any RNA can be used in the methods of the present invention, providedthat it has sufficient homology to the HDAC2 gene to mediate RNAi. TheRNA for use in the present invention can correspond to the entire HDAC2gene or a portion thereof. There is no upper limit on the length of theRNA that can be used. For example, the RNA can range from about 21 basepairs (bp) of the gene to the full length of the gene or more. Incertain embodiments the preferred length of the RNA of the invention is21 to 23 nucleotides.

Further, histone deacetylase DNA methylating enzymes can also beinhibited by binding peptides such as antibodies. Numerous histonedeacetylase antibodies are commercially available from sources such asSigma, Vinci Biochem, Cell Signaling Technologies. Such antibodies canbe modified to produce antibody fragments or humanized versions.Alternatively therapeutically useful antibodies can be produced usingtechniques known to those of ordinary skill in the art since HDACs areavailable.

The therapeutic compounds of the invention may be directly administeredto the subject or may be administered in conjunction with a deliverydevice or vehicle. Delivery vehicles or delivery devices for deliveringtherapeutic compounds to surfaces have been described. The therapeuticcompounds of the invention may be administered alone (e.g., in saline orbuffer) or using any delivery vehicles known in the art. For instancethe following delivery vehicles have been described: Cochleates;Emulsomes, ISCOMs; Liposomes; Live bacterial vectors (e.g., Salmonella,Escherichia coli, Bacillus calmatte-guerin, Shigella, Lactobacillus);Live viral vectors (e.g., Vaccinia, adenovirus, Herpes Simplex);Microspheres; Nucleic acid vaccines; Polymers; Polymer rings;Proteosomes; Sodium Fluoride; Transgenic plants; Virosomes; Virus-likeparticles. Other delivery vehicles are known in the art and someadditional examples are provided below.

The term effective amount of a therapeutic compound of the inventionrefers to the amount necessary or sufficient to realize a desiredbiologic effect. For example, as discussed above, an effective amount ofa therapeutic compounds of the invention is that amount sufficient tore-establish access to a memory. Combined with the teachings providedherein, by choosing among the various active compounds and weighingfactors such as potency, relative bioavailability, patient body weight,severity of adverse side-effects and preferred mode of administration,an effective prophylactic or therapeutic treatment regimen can beplanned which does not cause substantial toxicity and yet is entirelyeffective to treat the particular subject. The effective amount for anyparticular application can vary depending on such factors as the diseaseor condition being treated, the particular therapeutic compounds beingadministered the size of the subject, or the severity of the disease orcondition. One of ordinary skill in the art can empirically determinethe effective amount of a particular therapeutic compounds of theinvention without necessitating undue experimentation. Compositions ofthe invention include compounds as described herein, or apharmaceutically acceptable salt or hydrate thereof.

Subject doses of the compounds described herein for delivery typicallyrange from about 0.1 μg to 10 mg per administration, which depending onthe application could be given daily, weekly, or monthly and any otheramount of time therebetween. The doses for these purposes may range fromabout 10 μg to 5 mg per administration, and most typically from about100 μg to 1 mg, with 2-4 administrations being spaced days or weeksapart. In some embodiments, however, parenteral doses for these purposesmay be used in a range of 5 to 10,000 times higher than the typicaldoses described above.

In one embodiment, the composition is administered once daily at a doseof about 200-600 mg. In another embodiment, the composition isadministered twice daily at a dose of about 200-400 mg. In anotherembodiment, the composition is administered twice daily at a dose ofabout 200-400 mg intermittently, for example three, four, or five daysper week. In another embodiment, the composition is administered threetimes daily at a dose of about 100-250 mg. In one embodiment, the dailydose is 200 mg, which can be administered once-daily, twice-daily, orthree-times daily. In one embodiment, the daily dose is 300 mg, whichcan be administered once-daily or twice-daily. In one embodiment, thedaily dose is 400 mg, which can be administered once-daily ortwice-daily. The HDAC inhibitor can be administered in a total dailydose of up to 800 mg once, twice or three times daily, continuously(i.e., every day) or intermittently (e.g., 3-5 days a week).

For any compound described herein the therapeutically effective amountcan be initially determined from animal models. A therapeuticallyeffective dose can also be determined from human data for HDACinhibitors which have been tested in humans (e.g. for the treatment ofcancer) and for compounds which are known to exhibit similarpharmacological activities. Higher doses may be required for parenteraladministration. The applied dose can be adjusted based on the relativebioavailability and potency of the administered compound. Adjusting thedose to achieve maximal efficacy based on the methods described aboveand other methods as are well-known in the art is well within thecapabilities of the ordinarily skilled artisan.

Toxicity and efficacy of the prophylactic and/or therapeutic protocolsof the present invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Prophylacticand/or therapeutic agents that exhibit large therapeutic indices arepreferred. While prophylactic and/or therapeutic agents that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein.

Multiple doses of the molecules of the invention are also contemplated.In some instances, when the molecules of the invention are administeredwith another therapeutic a sub-therapeutic dosage of either agent, or asub-therapeutic dosage of both, is used. A “sub-therapeutic dose” asused herein refers to a dosage which is less than that dosage whichwould produce a therapeutic result in the subject if administered in theabsence of the other agent. Thus, the sub-therapeutic dose of, forinstance, an anti-Alzheimer's agent is one which would not produce thedesired therapeutic result in the subject in the absence of theadministration of the compounds of the invention.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, and optionally other therapeutic ingredients.

For use in therapy, an effective amount of the therapeutic compounds ofthe invention can be administered to a subject by any mode that deliversthe therapeutic agent or compound to the desired surface, e.g., mucosal,systemic. Administering the pharmaceutical composition of the presentinvention may be accomplished by any means known to the skilled artisan.Preferred routes of administration include but are not limited to oral,parenteral, intramuscular, intranasal, sublingual, intratracheal,inhalation, ocular, vaginal, rectal and intracerebroventricular.

For oral administration, the therapeutic compounds of the invention canbe formulated readily by combining the active compound(s) withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.Pharmaceutical preparations for oral use can be obtained as solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. Optionally the oral formulations may also be formulated insaline or buffers, i.e. EDTA for neutralizing internal acid conditionsor may be administered without any carriers.

Also specifically contemplated are oral dosage forms of the abovecomponent or components. The component or components may be chemicallymodified so that oral delivery of the derivative is efficacious.Generally, the chemical modification contemplated is the attachment ofat least one moiety to the component molecule itself, where said moietypermits (a) inhibition of proteolysis; and (b) uptake into the bloodstream from the stomach or intestine. Also desired is the increase inoverall stability of the component or components and increase incirculation time in the body. Examples of such moieties include:polyethylene glycol, copolymers of ethylene glycol and propylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline. Abuchowski and Davis, 1981, “SolublePolymer-Enzyme Adducts” In: Enzymes as Drugs, Hocenberg and Roberts,eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark, et al.,1982, J. Appl. Biochem. 4:185-189. Other polymers that could be used arepoly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

The location of release may be the stomach, the small intestine (theduodenum, the jejunum, or the ileum), or the large intestine. Oneskilled in the art has available formulations which will not dissolve inthe stomach, yet will release the material in the duodenum or elsewherein the intestine. Preferably, the release will avoid the deleteriouseffects of the stomach environment, either by protection of thetherapeutic agent or by release of the biologically active materialbeyond the stomach environment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is important. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused.

The shell material of cachets could be thick starch or other ediblepaper. For pills, lozenges, molded tablets or tablet triturates, moistmassing techniques can be used.

The therapeutic can be included in the formulation as finemulti-particulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, thetherapeutic agent may be formulated (such as by liposome or microsphereencapsulation) and then further contained within an edible product, suchas a refrigerated beverage containing colorants and flavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential non-ionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the therapeutic agenteither alone or as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery of the therapeuticcompounds of the invention. The therapeutic agent is delivered to thelungs of a mammal while inhaling and traverses across the lungepithelial lining to the blood stream. Other reports of inhaledmolecules include Adjei et al., 1990, Pharmaceutical Research,7:565-569; Adjei et al., 1990, International Journal of Pharmaceutics,63:135-144 (leuprolide acetate); Braquet et al., 1989, Journal ofCardiovascular Pharmacology, 13(suppl. 5):143-146 (endothelin-1);Hubbard et al., 1989, Annals of Internal Medicine, Vol. III, pp. 206-212(a1-antitrypsin); Smith et al., 1989, J. Clin. Invest. 84:1145-1146(a-1-proteinase); Oswein et al., 1990, “Aerosolization of Proteins”,Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,Colorado, March, (recombinant human growth hormone); Debs et al., 1988,J. Immunol. 140:3482-3488 (interferon-g and tumor necrosis factor alpha)and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colonystimulating factor). A method and composition for pulmonary delivery ofdrugs for systemic effect is described in U.S. Pat. No. 5,451,569,issued Sep. 19, 1995 to Wong et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but to not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colorado; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of therapeutic agent. Typically, each formulation is specificto the type of device employed and may involve the use of an appropriatepropellant material, in addition to the usual diluents, and/or carriersuseful in therapy. Also, the use of liposomes, microcapsules ormicrospheres, inclusion complexes, or other types of carriers iscontemplated. Chemically modified therapeutic agent may also be preparedin different formulations depending on the type of chemical modificationor the type of device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise therapeutic agent dissolved in waterat a concentration of about 0.1 to 25 mg of biologically active compoundper mL of solution. The formulation may also include a buffer and asimple sugar (e.g., for stabilization and regulation of osmoticpressure). The nebulizer formulation may also contain a surfactant, toreduce or prevent surface induced aggregation of the compound caused byatomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the therapeutic agentsuspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing therapeutic agent and may alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, or mannitolin amounts which facilitate dispersal of the powder from the device,e.g., 50 to 90% by weight of the formulation. The therapeutic agentshould most advantageously be prepared in particulate form with anaverage particle size of less than 10 mm (or microns), most preferably0.5 to 5 mm, for most effective delivery to the distal lung.

Nasal delivery of a pharmaceutical composition of the present inventionis also contemplated. Nasal delivery allows the passage of apharmaceutical composition of the present invention to the blood streamdirectly after administering the therapeutic product to the nose,without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran.

For nasal administration, a useful device is a small, hard bottle towhich a metered dose sprayer is attached. In one embodiment, the metereddose is delivered by drawing the pharmaceutical composition of thepresent invention solution into a chamber of defined volume, whichchamber has an aperture dimensioned to aerosolize and aerosolformulation by forming a spray when a liquid in the chamber iscompressed. The chamber is compressed to administer the pharmaceuticalcomposition of the present invention. In a specific embodiment, thechamber is a piston arrangement. Such devices are commerciallyavailable.

Alternatively, a plastic squeeze bottle with an aperture or openingdimensioned to aerosolize an aerosol formulation by forming a spray whensqueezed is used. The opening is usually found in the top of the bottle,and the top is generally tapered to partially fit in the nasal passagesfor efficient administration of the aerosol formulation. Preferably, thenasal inhaler will provide a metered amount of the aerosol formulation,for administration of a measured dose of the drug.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,1990, which is incorporated herein by reference.

The therapeutic compounds of the invention and optionally othertherapeutics may be administered per se (neat) or in the form of apharmaceutically acceptable salt. When used in medicine the salts shouldbe pharmaceutically acceptable, but non-pharmaceutically acceptablesalts may conveniently be used to prepare pharmaceutically acceptablesalts thereof. Such salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic,tartaric, citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The pharmaceutical compositions of the invention contain an effectiveamount of a therapeutic compound of the invention optionally included ina pharmaceutically-acceptable carrier. The termpharmaceutically-acceptable carrier means one or more compatible solidor liquid filler, diluents or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm carrier denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being commingled with the compounds of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficiency.

The therapeutic agents may be delivered to the brain using a formulationcapable of delivering a therapeutic agent across the blood brainbarrier. One obstacle to delivering therapeutics to the brain is thephysiology and structure of the brain. The blood-brain barrier is madeup of specialized capillaries lined with a single layer of endothelialcells. The region between cells is sealed with a tight junction, so theonly access to the brain from the blood is through the endothelialcells. The barrier allows only certain substances, such as lipophilicmolecules through and keeps other harmful compounds and pathogens out.Thus, lipophilic carriers are useful for delivering non-lipohiliccompounds to the brain. For instance, DHA, a fatty acid naturallyoccurring in the human brain has been found to be useful for deliveringdrugs covalently attached thereto to the brain (Such as those describedin U.S. Pat. No. 6,407,137). U.S. Pat. No. 5,525,727 describes adihydropyridine pyridinium salt carrier redox system for the specificand sustained delivery of drug species to the brain. U.S. Pat. No.5,618,803 describes targeted drug delivery with phosphonate derivatives.U.S. Pat. No. 7,119,074 describes amphiphilic prodrugs of a therapeuticcompound conjugated to an PEG-oligomer/polymer for delivering thecompound across the blood brain barrier. The compounds described hereinmay be modified by covalent attachment to a lipophilic carrier orco-formulation with a lipophilic carrier. Others are known to those ofskill in the art.

The therapeutic agents of the invention may be delivered with othertherapeutics for enhancing memory retrieval or treating other symptomsor causes of disorders associated with the memory loss. For instance,environmental enrichment (EE) has been used for enhancing memories. EEinvolves creating a stimulating environment around a subject. Othertherapeutics may also be combined to treat the underlying disorder or toenhance memory recall.

Examples of combinations of the compounds of the present invention withother drugs in either unit dose or kit form include combinations with:anti-Alzheimer's agents, beta-secretase inhibitors, gamma-secretaseinhibitors, HMG-CoA reductase inhibitors, NSAID's including ibuprofen,N-methyl-D-aspartate (NMDA) receptor antagonists, such as memantine,cholinesterase inhibitors such as galantamine, rivastigmine, donepezil,and tacrine, vitamin E, CB-1 receptor antagonists or CB-1 receptorinverse agonists, antibiotics such as doxycycline and rifampin,anti-amyloid antibodies, or other drugs that affect receptors or enzymesthat either increase the efficacy, safety, convenience, or reduceunwanted side effects or toxicity of the compounds of the presentinvention. The compounds of the invention may also be delivered in acocktail of multiple HDAC inhibitors. The foregoing list of combinationsis illustrative only and not intended to be limiting in any way.

The invention also includes articles, which refers to any one orcollection of components. In some embodiments the articles are kits. Thearticles include pharmaceutical or diagnostic grade compounds of theinvention in one or more containers. The article may includeinstructions or labels promoting or describing the use of the compoundsof the invention.

As used herein, “promoted” includes all methods of doing businessincluding methods of education, hospital and other clinical instruction,pharmaceutical industry activity including pharmaceutical sales, and anyadvertising or other promotional activity including written, oral andelectronic communication of any form, associated with compositions ofthe invention in connection with treatment of cognitive disorders suchas Alzheimer's disease.

“Instructions” can define a component of promotion, and typicallyinvolve written instructions on or associated with packaging ofcompositions of the invention. Instructions also can include any oral orelectronic instructions provided in any manner.

Thus the agents described herein may, in some embodiments, be assembledinto pharmaceutical or diagnostic or research kits to facilitate theiruse in therapeutic, diagnostic or research applications. A kit mayinclude one or more containers housing the components of the inventionand instructions for use. Specifically, such kits may include one ormore agents described herein, along with instructions describing theintended therapeutic application and the proper administration of theseagents. In certain embodiments agents in a kit may be in apharmaceutical formulation and dosage suitable for a particularapplication and for a method of administration of the agents.

The kit may be designed to facilitate use of the methods describedherein by physicians and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the invention. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflects approval by the agency of manufacture,use or sale for human administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container.

The kit may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, ora similar pouch or tray form, with the accessories loosely packed withinthe pouch, one or more tubes, containers, a box or a bag. The kit may besterilized after the accessories are added, thereby allowing theindividual accessories in the container to be otherwise unwrapped. Thekits can be sterilized using any appropriate sterilization techniques,such as radiation sterilization, heat sterilization, or othersterilization methods known in the art. The kit may also include othercomponents, depending on the specific application, for example,containers, cell media, salts, buffers, reagents, syringes, needles, afabric, such as gauze, for applying or removing a disinfecting agent,disposable gloves, a support for the agents prior to administration etc.

The compositions of the kit may be provided as any suitable form, forexample, as liquid solutions or as dried powders. When the compositionprovided is a dry powder, the powder may be reconstituted by theaddition of a suitable solvent, which may also be provided. Inembodiments where liquid forms of the composition are sued, the liquidform may be concentrated or ready to use. The solvent will depend on thecompound and the mode of use or administration. Suitable solvents fordrug compositions are well known and are available in the literature.The solvent will depend on the compound and the mode of use oradministration.

The kits, in one set of embodiments, may comprise a carrier means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, tubes, and the like, each of the container meanscomprising one of the separate elements to be used in the method. Forexample, one of the containers may comprise a positive control for anassay. Additionally, the kit may include containers for othercomponents, for example, buffers useful in the assay.

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. In thecase of dosage forms suitable for parenteral administration the activeingredient is sterile and suitable for administration as a particulatefree solution. In other words, the invention encompasses both parenteralsolutions and lyophilized powders, each being sterile, and the latterbeing suitable for reconstitution prior to injection. Alternatively, theunit dosage form may be a solid suitable for oral, transdermal, topicalor mucosal delivery.

In a preferred embodiment, the unit dosage form is suitable forintravenous, intramuscular or subcutaneous delivery. Thus, the inventionencompasses solutions, preferably sterile, suitable for each deliveryroute.

In another preferred embodiment, compositions of the invention arestored in containers with biocompatible detergents, including but notlimited to, lecithin, taurocholic acid, and cholesterol; or with otherproteins, including but not limited to, gamma globulins and serumalbumins. More preferably, compositions of the invention are stored withhuman serum albumins for human uses, and stored with bovine serumalbumins for veterinary uses.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures and other monitoring information.

More specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i.v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material. The invention also provides anarticle of manufacture comprising packaging material, such as a box,bottle, tube, vial, container, sprayer, insufflator, intravenous (i.v.)bag, envelope and the like; and at least one unit dosage form of eachpharmaceutical agent contained within said packaging material. Theinvention further provides an article of manufacture comprisingpackaging material, such as a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; andat least one unit dosage form of each pharmaceutical agent containedwithin said packaging material. The invention further provides anarticle of manufacture comprising a needle or syringe, preferablypackaged in sterile form, for injection of the formulation, and/or apackaged alcohol pad.

In a specific embodiment, an article of manufacture comprises packagingmaterial and a pharmaceutical agent and instructions contained withinsaid packaging material, wherein said pharmaceutical agent is a HDAC2inhibitor and a pharmaceutically acceptable carrier, and saidinstructions indicate a dosing regimen for preventing, treating ormanaging a subject with cognitive disorders such as Alzheimer's disease.

Therapeutic Monitoring: The adequacy of the treatment parameters chosen,e.g. dose, schedule, adjuvant choice and the like, is determined byconventional methods for monitoring memory. In addition, the clinicalcondition of the patient can be monitored for the desired effect, e.g.increases in cognitive function. If inadequate effect is achieved thenthe patient can be boosted with further treatment and the treatmentparameters can be modified, such as by increasing the amount of thecomposition of the invention and/or other active agent, or varying theroute of administration.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The Examples,data and Figures of U.S. patent application Ser. No. 11/998,834 as wellas U.S. Provisional Patent Application 61/119,698, both of overlappinginventorship are hereby incorporated by reference.

EXAMPLES Methods

Environmental enrichment: Up to four mice were continuously housed in acage that contained two wheels for voluntary running and a variety oftoys (obtained form from Petco) to create tunnels, and climbing devices.Food and water was ad libitum. The food was hidden within the bedding.Toys and running wheels were changed on a daily basis.

Cannulation and injection: Microcannula were inserted into the lateralbrain ventricles. Sodiumbutyrate (Sigma; St. Louis, Mo.) was dissolvedin artificial cerebrospinal fluid (aCSF). A stock solution of TSA(Sigma) was dissolved in DMSO and diluted with aCSF before injection.

Generation of HDAC overexpression animals The mouse HDAC1 or HDAC2coding sequence was placed into exon 1 of the Tau gene, in-frame withthe endogenous initiation codon, thereby creating a fusion protein thatcontains the first 31 amino acids of Tau. HDAC2 KO was produced in thelaboratory of R.A.D. and engineered to contain loxP recombination sitessuch that Cre-mediated recombination deletes exons 5 and 6 which encodesthe key catalytic core of the HDAC protein.

Chemical delivery Sodium butyrate (sigma) was dissolved in saline. HDACinhibitors were dissolved in DMSO in 50 mg/ml and diluted with salineimmediate before injection (100 ul-150 ul, i.p.).

Immunoblotting and staining Lysates for immunoblotting were prepared asdescribed herein (see also Fischer, A. et al. Recovery of learning andmemory is associated with chromatin remodeling. Nature 447 (7141),178-182 (2007).). Briefly, to isolate histones, brain tissue washomogenized in TX-buffer (50 mM Tris HCl, 150 mM NACl, 2 mM EDTA, 1%Triton-100) and incubated at 4° C. for 15 min before centrifugation at2,000 r.p.m. (400 g) for 10 min. After a wash-step in TX-buffer thepellet was dissolved in TX-buffer containing 0.2 M HCl and incubated onice for 30 min, before a second centrifugation at 10,000 r.p.m. (9,300g) for 10 min. The supernatant was used for immunoblotting. Immunoblotdata were quantified by measuring the band intensity using NIH imagingsoftware and UN-SCAN-it gel digitizing software (Silk Scientific).Immunostaining was performed as described herein (see also Fischer, A.et al. Recovery of learning and memory is associated with chromatinremodeling. Nature 447 (7141), 178-182 (2007).) using LSMetal0 softwareand a confocal microscope (Zeiss).

Gene targeting construct for HDAC1 overexpression (OE) mice. The ˜1200nt-long mouse HDAC1 cDNA was amplified from a brain cDNA library andconfirmed by sequencing. The cDNA was then cloned upstream of thepolyadenylation (pA) signal of pC8N2 with a SpeI blunt ligation,subsequently HDAC1-pA was cloned into pBSK (Stratagene). A pGKneoLoxPsequence was directionally inserted into the XhoI-Kpn1 site downstreamof the HDAC1-pA in pBSK. The HDAC1-pA-neo was released with XmaI-Acc65and cloned in frame into exon 1 of the Tau gene. The Tau targeting armswere taken from pTauKR and modified by insertion of a XmaI and BsiWIlinker in the unique NcoI site. The resulting targeting vector (pTH1)containing the in frame fusion of HDAC1 coding sequence with exon 1 ofTau was confirmed by sequencing. 3-6-month-old mice were used for thebehavior test and further analysis.

Gene targeting construct for HDAC2 overexpression (OE) mice The mouseHDAC2 cDNA was obtained using RT PCR from mouse brain tissue. It wassequenced and subcloned into the XhoI-EcoR1 site of the Topo-TA vector(Invitrogen). The pTH1 targeting vector (described above) was cut openwith SmaI-SalI to release HDAC1. The HDAC2 cDNA was cut out from Topo-TAwith an EcoRI-XhoI and cloned into the SmaI-SalI site of pTH1, to createthe pTH2 targeting vector. The in frame fusion of HDAC2 to exon 1 of Tauwas verified by sequencing of pTH2.

The targeting vectors pTH1 and pTH2 were linearized with SacI andelectroporated into V6.5 (129XC57BL/6) F1 embryonic stem (ES) cell line.We picked 96 neomycin resistant clones, of which 46 were analyzed bysouthern blots. We only used a 3′ external probe, after digestion withBamHI (Left) and EcoRI (Right). Wild-type clones display a 8.8-kb band.The correct targeting event results in a band-shift to 13 kb for thetargeted allele. 5 clones were correctly targeted. Two clones were usedto generate chimeras by injections into (DBA/2XC57BL/6) F1 blastocysts.Chimeras were mated to C57BL/6 females and offspring was analyzed forgermline transmission. The heterozygous knock-in strains were maintainedin a mixed background and were mated to obtain homozygous animals.3-6-month-old mice were used for the behavior test and further analysis.

Generation of Hdac2 KO mice The Hdac2 floxed allele was generated byflanking exon 5 and exon 6 with loxP recombination sites, assuring thedeletion of the HDAC-catalytic core of the protein after Cre-recombinasemediated deletion. Upon successful targeting of ES-cells and subsequentderivation of chimeric mice, we established a mouse strain carrying afoxed allele of Hdac2 (Hdac2^(L))(FVB). Infection of mouse embryonicfibroblasts with retroviruses expressing Cre-recombinase resulted incomplete ablation of Hdac2 only in MEFs carrying two Hdac2 floxedalleles. This indicates that the floxed Hdac2 allele is functional andresults in an Hdac2 null-genotype upon Cre-recombinase expression.Deletion of Hdac2 in the germline using EIIa-Cre or Nestin-Cretransgenic mice resulted in viable and fertile Hdac2^(−/−) mice with noobvious histological abnormalities up to a year of age. CrossingHdac2^(+/−) mice gave rise to viable Hdac2-deficient mice, but thesemice were born with a 2-fold lower frequency than expected from a normalMendelian ratio (9 Hdac2^(−/−) mice out of 79 littermates, versus 20 outof 79 expected. Although Hdac2^(−/−) mice are viable and are capable ofproducing offspring their fertility is compromised (data not shown).Hdac2^(−/−) mice (males and females) were approximately 25% smallercompared to wild-type and heterozygote littermates (data not shown). Theanimals used for behavior tests are in FVBxC57/BL6 background and matedto each other to obtain homozygous animals. 3-6-month-old mice were usedfor the behavior test and further analysis. There was no difference inbehavior tests between males and females.

Fear conditioning tests Context-dependent fear conditioning. Trainingconsists of a 3 min exposure of mice to the conditioning box (context)followed by a foot shock (2 sec, 0.5/0.8/1.0 mA, constant current). Thememory test was performed 24 hr later by re-exposing the mice for 3 mininto the conditioning context. Freezing, defined as a lack of movementexcept for heart beat and respiration associated with a crouchingposture, was recorded every 10 sec by two trained observers (one wasunaware of the experimental conditions) during 3 min (a total of 18sampling intervals). The number of observations indicating freezingobtained as a mean from both observers was expressed as a percentage ofthe total number of observations.

For short time memory test, the memory test was performed 3 hrs afterthe foot shock training.

Tone-dependent fear conditioning. Training consisted of a 3 min exposureof mice to the conditioning box (context), followed by a tone [30 sec,20 kHz, 75 dB sound pressure level (SPL)] and a foot shock (2 sec, 0.8mA, constant current). The memory test was performed 24 hr later byexposing the mice for 3 min to a novel context followed by an additional3 min exposure to a tone (10 kHz, 75 dB SPL). Freezing was recordedevery 10 sec by two nonbiased observers as described above.

Morris water maze test The water maze paradigm was performed in acircular tank (diameter 1.8 m) filled with opaque water. A platform(11×11 cm) was submerged below the water's surface in the center of thetarget quadrant. The swimming path of the mice was recorded by a videocamera and analyzed by the Videomot 2 software (TSE). For each trainingsession, the mice were placed into the maze consecutively from fourrandom points of the tank. Mice were allowed to search for the platformfor 60 s. If the mice did not find the platform within 60 s, they weregently guided to it. Mice were allowed to remain on the platform for 15s. Two training trials were given every day; the latency for each trialwas recorded for analysis. During the memory test (probe test), theplatform was removed from the tank, and the mice were allowed to swim inthe maze for 60 s.

Spatial working memory on elevated T-maze Mice were maintained on arestricted feeding schedule at 85% of their free-feeding weight. Spatialworking memory was first assessed on an elevated plastic T-maze. Thisconsisted of a start arm (47×10 cm) and two identical goal arms (35×10cm), surrounded by a 10 cm high wall. A plastic food well was located 3cm from the end of each goal arm. The maze was located 1 m above thefloor in a well lit laboratory that contained various prominent distalextramaze cues. The mice were habituated to the maze, and to drinkingsweetened, condensed milk, over several days before spatialnon-matching-to-place testing.

Each trial consisted of a sample run and a choice run. On the samplerun, the mice were forced either left or right by the presence of aplastic block, according to a pseudorandom sequence (with equal numbersof left and right turns per session, and with no more than twoconsecutive turns in the same direction). A reward consisting of 0.07 mlof sweetened, condensed milk (diluted 50/50 with water) was available inthe food well at the end of the arm. The block was then removed, and themouse was placed, facing the experimenter, at the end of the start armand allowed a free choice of either arm. The time interval between thesample run and the choice run was approximately 15 s. The animal wasrewarded for choosing the previously unvisited arm (that is, foralternating). Mice were run one trial at a time with an inter-trialinterval (ITI) of approximately 10 min. Each daily session consisted of4 trials, and mice received 24 trials in total.

Chemical administration Suberoylanilide hydroxamic acid (SAHA) wassynthesized as described previously in WO 93/07148 PTC/US92/08454.Sodium butyrate was purchased from Sigma (cat.B5887). SAHA and WT-161were dissolved in DMSO as stock solutions and diluted in saline justbefore injection. Sodium butyrate was prepared in saline. Mice receivedintraperitoneal injection daily with either SAHA or saline for 10 daysor 21 days.

Golgi impregnation Golgi-Cox-stained brains were cut to 200 μm thickcross-sections with vibratome and analyzed using a Zeiss 200 Axiovertmicroscope and Openlab software. The number of apical and basal spineson hippocampal CA1 pyramidal neurons was counted blind to the genotype.For each experimental group, a minimum of 10 cells per slice (animalnumber n=3) were analyzed. CA1 hippocampal neurons within the region−1.4 mm to −1.6 mm (relative to the bregma position) were included forthe analysis.

Virus mediated spine labeling. Tomato expressing HSV (0.5 μl, gift fromRachael Neve) was stereo-injected into both sides of area CA 1 ordentate gyrus with 0.05 μl/min rate. Mice were sacrificed 48 hrs afterinjection. Brains were fixed with 4% PFA and sectioned with vibratome(50 μm, Leica). Hippocampal slices were scanned with a confocalmicroscope. Obtained image stacks were reconstructed and analyzed usingimage J.

Immunohistochemistry Immunohistochemical analysis was performed asdescribed before (Guan, J. S., et al., Cell, 2005. 122(4): p. 619-31.).Antibodies were used in a 1:1000 concentration. Anti-HDAC1, andanti-HDAC2 antibodies were purchased from Abcam. Anti-Ac-lysine,anti-Ac-H4K5, anit-Ac-H4K12, anti-Ac-H3K16, anti-CREB, anti-AKT andanti-CaMKIIa antibodies were purchased from Cell Signaling.Anti-Ac-α-tubulin (K40), anti-actin and anti-synaptophysin (SVP-38)antibodies were purchased from Sigma. Anti-NR2A and anti-NR2B werepurchased from BD Biosciences. Anti-β-catenin, anti-EGR1, anti-c-FOS,anti-Brn1, anti-TLE4, anti-CDP, anti-ER81 and anti-GAPDH antibodies werepurchased from Santa Cruz. Anti-NeuN antibody was purchased fromChemicon. Confocal images (1 μm) were scanned and subjected tothree-dimensional reconstruction. LSMetal0 software (Zeiss) was used tocalculate the mean synaptophysin intensity. Brain sections with thestrongest intensity were scanned first. All other images included in theanalysis were scanned using the same settings. Staining was quantifiedusing LSMetal0 software (Zeiss).

Protein extraction and immunoblotting. The hippocampus and forebrainwere collected and lysed in RIPA buffer. The lysates were incubated for15 min on ice and centrifuged for 15 min at 15,000×g at 4° C. Thesupernatant was collected as cytosolic protein extract. The lysates weresubjected to 10% SDS-PAGE followed by immunoblotting.

Extraction of histone proteins. Hippocampus samples were collected andhomogenized in 400 μl TX-buffer (50 mM Tris-HCl, pH8.5, 5 mM sodiumbutyrate). The pellets were resuspended in 0.2M HCl/TX buffer andincubated on ice for 30 mins. Samples were spun down at 14000 rpm, thehistone containing supernatants were subjected to western analysis.

Electrophysiological analysis. 3-6 months old HDAC2OE, HDAC2KO or theirlittermates were killed by cervical dislocation, and hippocampi wererapidly dissected in iced oxygenated artificial CSF (ACSF). Transversehippocampal slices, 400 μm thick were placed in a chamber andcontinuously perfused with oxygenated ACSF. A bipolar stimulatingelectrodes (0.002-inch-diameter nichrome wire; A-M Systems) placed inthe stratum radiatum was used to elicit action potentials in CA3 axons.An ACSF-filled glass microelectrode with a resistance between 0.5 and 3MΩ was placed in the stratum radiatum region of CA1 and was used torecord the field excitatory post-synaptic potentials (fEPSP). Data wereacquired using HEKA EPC10 and analyzed by patchmaster (HEKA). Peak fEPSPamplitudes from stimulators were required to be at least 2 mV, andstimulus intensity was set to produce 40% of the maximal response.Baseline responses were recorded for 20 min. fEPSP were evoked at theCA1 synapses by stimulating Schaffer collaterals at a low frequency (2per min) to establish a stable baseline. Immediately following LTPinduction with high-frequency stimulation (HFS, 100 Hz, 1 s), slicesfrom HDAC2OE and control mice showed an increase in fEPSP slope andamplitude, suggesting that short-term potentiation (STP) occurs in allgroups. For HDAC2KO and its control WT slices, LTP was induced byapplying one train of stimuli at 100 Hz for 1 s. For HDAC2OE and itscontrol WT slices, LTP was induced by applying two trains of stimuli at100 Hz for 1 s, with an interval of 20 s.

Imaging based EGR-1 expression assay for cultured neurons Embryoniccortici (E17) of EGR1-GFP BAC transgenic mice (Genesat Project) wereisolated using standard procedures and triturated with trypsin/DNAsedigestion. Cortical neurons were plated at a density of 10,000 cells perwell in black/clear bottom plates coated with poly-D-lysine (Costar) inneurobasal medium (1.6% B27, 2% glutamax, 1% pen/strep and 5% heatinactivated fetal calf serum) and in neurobasal medium without serum 24hrs later. Under these culture conditions, the percentage of glia wasestimated to be in the range of 5-25. On day 6, HDAC inhibitors or DMSOcontrol (triplicates or quadruplicates) were added to the cultures forapprox. 30 hr. BDNF, KCl or forskolin were added to the cultures on day7 for 8 hrs.

Cell were fixed in 4% PFA/4% sucrose in PBS. Fixative was washed awaywith PBS (3 wash cycles) and processed for EGR1-GFPimaging. Cells(3,000-5,000 per well) were imaged and analyzed with 5× objective usingthe Cellomics ArrayScan Image system. The built-in TargetActivationalgorithm was optimized to measure average EGR1-GFP expression per cell(mean Fluorescence intensity per cell per well), using the Hoechst dyeto mark cells. The data was normalized to control (medium addition).

After imaging, cells were processed for antibody staining: cells werepermeabilized with 0.25% TritonX100 (10-15 min). Triton was washed awayby 3 PBS wash cycles, cells were blocked in PBS containing 10% goat orhorse serum (1 hr, 37° C.). Cells were exposed toanti-acetyl-Lysine-histone H3 or H4 antibody. Then washed 5 times withPBS followed by secondary antibody conjugated to Alexa594, and Hoechst(1 hr, RT). Secondary antibody was washed 5 times with PBS, and assayedon Cellomics ArrayScan Image system.

Chromatin immunoprecipitation (ChIP) ChIP was performed using mouseforebrains fixed with 4% PFA solution and stored at −80° C. prior touse. Brains were chemically cross-linked by the addition of one-tenthvolume of fresh 11% formaldehyde solution for 15 min at roomtemperature, homogenized, resuspended, lysed in lysis buffers, andsonicated to solubilize and shear crosslinked DNA. Sonication conditionsvary depending on cells, culture conditions, crosslinking, andequipment. We used a Misonix Sonicator 3000 and sonicated at power 7 for10×30 s pulses (90 s pause to between pulses) at 4° C. while sampleswere immersed in an ice bath. The resulting whole-cell extract wasincubated overnight at 4° C. with 100 μl of Dynal Protein G magneticbeads that had been preincubated with 10 μg of the appropriate antibody.Beads were washed five times with RIPA buffer and one time with TEcontaining 50 mM NaCl. Bound complexes were eluted from the beads byheating at 65° C. with occasional vortexing and crosslinking wasreversed by overnight incubation at 65° C. Whole-cell extract DNA(reserved from the sonication step) was also treated for crosslinkreversal. Immunoprecipitated DNA and whole-cell extract DNA were thenpurified by treatment with RNaseA, proteinase K, and multiplephenol:chloroform:isoamyl alcohol extractions. Purified DNA samples werenormalized and subjected to PCR analysis. Antibodies used for pull downswere: anti-HDAC1 (#31263), anti-HDAC2(#12169) from Abcam; anti-ACH4(#06-866), anti-ACH3 (#06-599) from Upstate. After IP, recoveredchromatin fragments were subjected to semiquantitative PCR or Real-timePCR for 32-40 cycles using primer pairs specific for 150-250 bp segmentscorresponding to mouse genes promoter regions (regions upstream of thestart codon, near the first exon).

Real-time PCR: Real-time PCR was carried out with SYBR-Green-basedreagents (Invitrogen, express SYBR GreenER) using a CFX96 real-time PCRDetection system (BioRad). The relative quantities of immunoprecipitatedDNA fragments were calculated using the comparative C_(T) method.Results were compared to a standard curve generated by serial dilutionsof input DNA. Data were derived from three independent amplifications.Error bars represent standard deviations.

Primer sequences used for PCR:

BDNF PI: (SEQ ID NO: 3) 5′-TGATCATCACTCACGACCACG-3′ (SEQ ID NO: 4)5′-CAGCCTCTCTGAGCCAGTTACG-3′ BDNF PII: (SEQ ID NO: 5)5′-TGAGGATAGTGGTGGAGTTG-3′ (SEQ ID NO: 6) 5′-TAACCTTTTCCTCCTCC-3′BDNF PIV: (SEQ ID NO: 7) 5′-GCGCGGAATTCTGATTCTGGTAAT-3′ (SEQ ID NO: 8)5′GAGAGGGCTCCACGCTGCCTTGACG-3′ CREB: (SEQ ID NO: 9)5′-CTACACCAGCTTCCCCGGT-3′ (SEQ ID NO: 10) 5′-ACGGAAACAGCCGAGCTC-3PKM zeta (100 bp upstream of the PKMzeta mRNAinitiation site [15], which contains acAMP response element (CRE) consensus sequence): (SEQ ID NO: 11)5′-TGTTGAGTCTGGGCCCTC-3′ (SEQ ID NO: 12) 5′-CCTGGCCTCCGGACC-3′Creb binding protein (CBP): (SEQ ID NO: 13) 5′-CGGGCAGGGGATGAG-3′(SEQ ID NO: 14) 5′-GCGAGCCAGCGAGGA-3′ Neurexin I: (SEQ ID NO: 15)5′-CAGGGCCTTTGTCCTGAATA-3′ (SEQ ID NO: 16) 5′-GCTTTGAATGGGGTTTTGAG-3′Neurexin III: (SEQ ID NO: 17) 5′-ACTGAGAGCTAGCCACCCAGAC-3′(SEQ ID NO: 18) 5′-TTGCCCATTTGTGAATTTGA-3′ PGK1: (SEQ ID NO: 19)5′-ACATTTTGGCAACACCGRGAG-3′ (SEQ ID NO: 20)5′-GAAGTAGCACGTCTCACTAGTCTCGTG-3′ ATF4: (SEQ ID NO: 21)5′-GTGATAACCTGGCAGCTTCG-3′ (SEQ ID NO: 22) 5′-GGGGTAACTGTGGCGTTAGA-3′CaMKIIA: (SEQ ID NO: 23) 5′-GACCTGGATGCTGACGAAG-3′ (SEQ ID NO: 24)5′-AGGTGATGGTAGCCATCCTG-3′ p21 (WAP/CIP1): (SEQ ID NO: 25)5′-CCACAGTTGGTCAGGGACAG-3′ (SEQ ID NO: 26) 5′-CCCTCCCCTCTGGGAATCTA-3′EGR-1: (SEQ ID NO: 27) 5′-GTGCCCACCACTCTTGGAT-3′ (SEQ ID NO: 28)5′-CGAATCGGCCTCTATTTCAA-3′ Agrin: (SEQ ID NO: 29)5′-TTGTAACCAACAGGGGTTGC-3′ (SEQ ID NO: 30) 5′-AGTTGTGGCTAGGGGAGCAC-3′EGR-2: (SEQ ID NO: 31) 5′-GGCTGCAAATCGTTCCTG-3′ (SEQ ID NO: 32)5′-TCGGAGTATTTATGGGCAGGT-3′GLUTAMATE RECEPTOR 1 PRECURSOR (GLUR-1/AMPA 1) (SEQ ID NO: 33)5′-GGAGGAGAGCAGAGGGAGAG-3′ (SEQ ID NO: 34) 5′-TTCCTGCAATTCCTTGCTTG-3′GLUR-2 (SEQ ID NO: 35) 5′-GCGGTGCTAAAATCGAATGC-3′ (SEQ ID NO: 36)5′-ACAGAGAGGGGCAGGCAGT-3′ PSD95: (SEQ ID NO: 37)5′-CCCCTACCCCTCCTGAGAAT-3′ (SEQ ID NO: 38) 5′-GAGGGGAAGGAGAAGGTTGG-3′HOMER1: (SEQ ID NO: 39) 5′-CTGCCTGAGTGTCGTGGAAG-3′ (SEQ ID NO: 40)3′-ATGATTTCACTCGCGCTGAC3′ P35: (SEQ ID NO: 41) 5′-GAGGGAGGGCGCTGAGG-3′(SEQ ID NO: 42) 5′-GCAGCTAGGGAGCTTCTGTCC-3′ CDK5: (SEQ ID NO: 43)5′-CGCAGCCTGTTGGACTTTGT-3′ (SEQ ID NO: 44) 3′-GCGTTGCAGAGGAGGTGGTA-3′SHANK3: (SEQ ID NO: 45) 5′-TTTTCCAGGTCCCAGTGGTG-3′ (SEQ ID NO: 46)5′-CCTGCCCACAGTGTCACTCC-3′ SVP: (SEQ ID NO: 47)5′-CTAGCCTCCCGAATGGAATG-3′ (SEQ ID NO: 48) 5′-CAGCAGCAGCATCAGCAATG-3′SYNAPSIN2 (SEQ ID NO: 49) 5′-GGCTTTCCTTCCCTCCACAC3′ (SEQ ID NO: 50)5′TGTTAGCGAGGGAGCAGTGG3′ BETA-ACTIN: (SEQ ID NO: 51)5′-CCCATCGCCAAAACTCTTCA3′ (SEQ ID NO: 52) 5′GGCCACTCGAGCCATAAAAG3′GAPDH: (SEQ ID NO: 53) 5′-CTCCCAGGAAGACCCTGCTT-3′ (SEQ ID NO: 54)5′-GGAACAGGGAGGAGCAGAGA-3′ ARC: (SEQ ID NO: 55)5′-CAGCATAAATAGCCGCTGGT-3′ (SEQ ID NO: 56) 5′-GAGTGTGGCAGGCTCGTC-3′ FOS:(SEQ ID NO: 57) 5′-GAAAGCCTGGGGCGTAGAGT-3′ (SEQ ID NO: 58)5′-CCTCAGCTGGCGCCTTTAT-3′ CPG15: (SEQ ID NO: 59)5′-GCGAGATTTCGTTGAGATCG-3′ (SEQ ID NO: 60) 5′-GGGATGACACGGATTGATTTT-3′SNK: (SEQ ID NO: 61) 5′-TTTCCCACGTCCAAAGTCAG-3′ (SEQ ID NO: 62)5′-GCAGCGAAGCTTTAAATACGC-3′ NR2A: (SEQ ID NO: 63)5′-TCGGCTTGGACTGATACGTG-3′ (SEQ ID NO: 64) 5′-AGGATAGACTGCCCCTGCAC-3′NR2B: (SEQ ID NO: 65) 5′-CCTTAGGAAGGGGACGCTTT-3′ (SEQ ID NO: 66)5′-GGCAATTAAGGGTTGGGTTC-3′ TUBULIN: (SEQ ID NO: 67)5′-TAGAACCTTCCTGCGGTCGT-3′ (SEQ ID NO: 66) 5-TTTTCTTCTGGGCTGGTCTC-3′

Statistical analysis: The data were analyzed by unpaired student's ttest and one-way ANOVA (ANalyis Of VAriance). One-way ANOVA followed bypost-hoc Scheffe's test was employed to compare means from severalgroups. Error bars present S.E.M.

Results

Example 1

SAHA was administered daily by intraperitoneal (i.p.) injection at 25mg/kg for 10 days prior to contextual fear conditioning training andmemory test. Remarkably, SAHA, but not saline treatment, significantlyincreased the freezing behavior of HDAC2OE mice (66.7±5.1%, n=12;26.9±5.9, n=12, p<0.0001, SAHA group versus saline group, FIG. 1A). Itshould be noted that, in the same training paradigms, SAHA treatmentincreased the freezing behavior of WT control mice from 44.8±4.7% (n=15,saline control) to 63.9±4.2% (n=12, SAHA treatment). Thus, the freezinglevels of HDAC2OE mice after SAHA treatment were comparable to those ofthe control mice treated with SAHA, despite the fact that saline treatedHDAC2OE mice exhibited lower freezing behavior. Concordantly, SARAtreatment completely abrogated the decreased dendritic spine andsynapses phenotype in HDAC2OE mice (FIG. 1B,C).

Next, we investigated the effect of SAHA on HDAC2KO mice. As HDAC2KOmice showed markedly increased freezing behavior compared to WTlittermates without treatment, we sensitized the assay by lowering thefoot shock intensity from 1.0 mA to 0.5 mA to prevent a possible ceilingeffect in the memory test. Using this paradigm, we found that SAHAtreatment (n=10) induced significantly higher freezing behavior(p=0.0383) compared to saline treatment (n=10) in the WT control mice(45.0±6.9% v.s. 25.0±5.8%, FIG. 1D). However, SAHA treatment did notalter the freezing behavior of the HDAC2KO mice compared to salinetreatment (52.1±9.8% v.s. 49.3±8.4%, p=0.8324, n=8 for each group) (FIG.1D). Furthermore, dendritic spine density of CA1 neurons andsynaptophysin staining in the stratum radiatum of the HDAC2KO mice wasnot significantly affected by SAHA treatment (FIG. 1E,F). Consistently,although SAHA treatment modestly increased LTP in the WT hippocampus, itdid not have a detectable effect on LTP in the HDAC2 KO hippocampus(FIG. 6). Thus, HDAC2 KO mice are refractory to synaptogenesis andfacilitation of synaptic plasticity and memory formation induced bySAHA. These results strongly suggest that HDAC2 is the major, if not theonly target of SAHA in eliciting memory enhancement.

SAHA was initially reported to be a pan-HDACi, although recent studiesusing recombinant HDACs and in vitro deacetylase assays with appropriateclass-specific substrates have revealed that SAHA is a more potentinhibitor of class I HDACs and HDAC6, with very weak to no inhibition ofclass IIa HDACs, such as HDAC4, 5, and 7. Although SB does not inhibitthe activity of HDAC6 in vitro, to directly address the potentialimportance of this class IIb HDAC, we tested whether selectivelyinhibiting HDAC6 has any effects on memory formation using the HDACiWT-161 (FIG. 2A,B). α-Tubulin(K40) deacetylation is a known non-histonesubstrate of HDAC6 that served as specificity control in theseexperiments. While WT-161 increased α-tubulin(K40) levels in hippocampalpyramidal neurons(FIG. 2C), there was no correlated increase in memoryformation (FIG. 2D). This result, and the observed cellular selectivityof SB and WT-161, suggests that HDAC6 inhibition by SAHA might not beinvolved in HDACi induced memory enhancement. In agreement with these,proteome-wide studies of a SAHA-based affinity probe identified HDAC1and HDAC2 as the main cellular targets. Thus, class I HDACs, especiallyHDAC1 and HDAC2, might be the potential target for HDACi induced memoryenhancement.

To directly evaluate the physiological role of HDAC1 and HDAC2 in thebrain, we generated two mouse lines in which HDAC1 or HDAC2 wasover-expressed in neurons. The mouse HDAC1 or HDAC2 coding sequence wasplaced into exon 1 of the Tau gene, in-frame with the endogenousinitiation codon, thereby creating a fusion protein that contains thefirst 31 amino acids of Tau. Previously, homozygous animals mutant forTau were shown to be phenotypically indistinguishable from wild-typelittermates in memory tests. A 2-3 fold increase in HDAC1 or HDAC2protein expression in brain of homozygous animals as compared to WT micewas observed in the hippocampus and other areas of the brain (FIG. 3).Consistently, the overall acetylated lysine level was reduced inhomozygous HDAC1 (HDAC1OE) and HDAC2 overexpression mice (HDAC2OE),especially in the pyramidal neurons of the hippocampal formation. Wefound acetylated H4K12, H4K5 but not H3K14 was decreased in brains ofHDAC2OE mice (data not shown). In contrast, acetylated α-tubulin(K40)level did not change in the HDAC1OE or HDAC2OE mice. Thus, the HDAC1/2overexpressing animals exhibited increased histone deacetylation in thebrain compared to that of the wildtype (WT) littermates. Importantly,there was no discernable difference in gross brain anatomy or neuronalpositioning in the HDAC1/2 overexpressing mice, suggesting thatincreased HDAC1/2 is not overtly detrimental to brain development orneuronal survival.

Western blots from brain lysate were performed and showed theup-regulation of HDAC1 and HDAC2 respectively in HDAC1 or HDAC2homozygous over-expression mice (data not shown). Decreased histoneacetylation in the hippocampus of HDAC1OE and HDAC2OE mice was observed.Samples from hippocampal histone preparation also showed the reductionof lysine acetylation (at ˜16 KDa) in HDAC1OE mice and HDAC2OE mice.

Interestingly, in the short-term memory test, no significant differencecould be detected among HDAC1OE, HDAC2OE and WT control in both thecontext- and tone-dependent fear learning 3 hours after training (FIG.4B). These observations suggest that HDAC2, but notHDAC1-gain-of-function in the nervous system results in impairment inassociative learning. The escape latency and swimming speed were notdifferent among groups in the visible platform test (FIGS. 4A&B),indicating comparable motor and visual function among the variousstrains. These results revealed a marked reduction of spatial learningof the HDAC2OE mice. Furthermore, HDAC2OE mice but not HDAC1OE miceshowed spatial working memory impairment in a T-maze non-matching-toplace task (FIG. 4H). Thus, gain of function of HDAC2, but not HDAC1,impairs hippocampus dependent memory formation.

The HDAC 2 gene knockout enhances associative learning. To furtherinvestigate the role of HDAC2 in associative learning, HDAC2 deficientmice (HDAC2KO) were generated, by crossing mice carrying a floxed Hdac2allele with Nestin-Cre transgenic mice. Germ-line deletion of Hdac2resulted in viable and fertile Hdac2^(+/−) mice with no obvioushistological abnormalities up to a year of age (FIG. 5). CrossingHdac2^(+/−) mice gave rise to viable Hdac2-deficient mice, in whichHDAC2 expression was abolished in the brain.

The freezing behavior of HDAC2 knockout (KO) mice and control mice(HDAC2 KO n=10; control, n=10) during the contextual dependent memorytest was examined. HDAC2 KO mice showed enhanced fear conditioning.

Loss of HDAC2 does not lead to detectable changes in the anatomy or cellpositioning in the brain. H4K5, H4K12 and H2B acetylation wassignificantly increased in the hippocampus of HDAC2KO mice. However,overall acetylation of lysine residues in histone preparation wasslightly decreased as revealed by western blot analysis using theacetylated-lysine antibody. This might be the consequence of acompensatory increase of HDAC1 in HDAC2KO mice (FIG. 5D). Remarkably,the HDAC2KO mice (n=9) showed markedly increased freezing behavior asevaluated by the contextual- and tone-dependent fear conditioningparadigm (p=0.0036, p=0.0047, FIG. 12A) 24 hours after training whencompared to WT littermates (n=11). In the short-term memory test,HDAC2KO mice (n=9) showed increased freezing behavior (p=0.010 FIG. 4E)comparing to WT littermates (n=8) in contextual dependent conditioning.No difference in the locomotor activity or pain sensation had beendetected between these two groups of mice. Thus, HDAC2 loss of functionenhanced associative learning. Furthermore, HDAC2KO mice showed aprofound spatial working memory improvement in the T-mazenon-matching-to place task (p=0.025, two-way ANOVA, FIG. 4G). Thesedata, coupled with the gain of function studies, suggest that HDAC2 maynegatively regulate memory formation in mice.

Example 2

In vitro assays were used to test the protective effects of HDACoverexpression on p25 induced toxicity. Neurons are dissociated fromE15.5 cortex and hippocampus. They were transfected with plasmidsencoding p25-GFP and Flag-HDACs at DIV4. 24 hrs after transfection,neurons were fixed and processed for IHC. HDAC1,5,6,7 and 10 showedprotection (FIG. 7).

In summary, using mouse genetic models, we delineated the functions ofHDAC isoforms including class I HDACs such as HDAC1 and HDAC2, andshowed evidence that HDAC2 plays a negative role in regulating memoryformation. Notably, we identified HDAC2 as the major target of HDACi infacilitating learning and memory. Our observations support the notionthat HDAC1 and HDAC2 differentially regulate subset of activityregulated genes or genes implicated in plasticity and memory. This isunexpected, given the fact that HDAC1 and HDAC2 were reported to formfunctional hetero-dimmers (Grozinger, C. M. & Schreiber, S. L. Chem Biol9 (1), 3-16 (2002)). It is possible that this is due to the differentialdistribution of HDAC1 and HDAC2 in the brain as described herein.Alternatively, neuronal HDAC2 and HDAC1 might form distinct complexeswith transcriptional co-repressors and therefore are enriched indifferent regions of the chromatin. Additionally, HDAC2 maydifferentially target additional non-histone proteins, which may beinvolved in memory formation. Other possibilities, such as difference inposttranscriptional modification might also contribute to thebiochemical/functional dissociation between HDAC1 and HDAC2. It shouldbe noted that HDAC1 deficiency in mice is detrimental, resulting inembryonic lethality. We have also discovered that HDAC1 loss of functionin neurons causes DNA damage and cell death. Conversely, HDAC2 deficientmice are viable and exhibit enhanced memory formation. These results notonly reveal important distinct functions of HDAC isoforms, and hence,their target genes or non-histone substrates, they also support thediscovery that HDAC2 is a suitable target for memory enhancement.

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Example 3 In Vitro Enzymatic Inhibitions Assay Data

The enzymatic inhibitory activity of multiple HDAC inhibitors wasassayed against several of the known HDAC isoforms and is shown in FIG.8. SAHA was included as a reference mixed class I-class II inhibitor.BRD-6929 demonstrates that this class of compounds does not inhibitHDAC8 or the Class II HDAC enzymes. All of the BRD numbered compoundsare derived from the ortho-anilide class of compounds. Not all compoundsfrom this class are expected to bind the class II HDACs.

Example 4 In Vitro Cellular Data in Non-Neuronal Cell Lines

Standard western blotting methods were used to measure the effects ofHDAC inhibitors on histone acetylation marks in HeLa cell lysate. Seriesof compounds were incubated with whole HEK293 cells at 10 uM for a 6hour time period. Western blot showed increased acetylation levels overDMSO controls using anti-acetyl H4K12 antibodies and horseradishperoxidase conjugated secondary antibody along with a luminol-basedsubstrate (FIG. 9). This demonstrates cellular HDAC activity of theseanalogs and the increase in acetylation in the specific mark, H4K12.Quantification of the raw western data (FIG. 10) established thatrelative to the DMSO control, multiple selectivity profiles areeffective in increasing H4K12 acetylation levels, and that HDAC 1,2 andHDAC 1,2,3 selective inhibitors have robust HDAC activity in whole cellson a specific histone loci (H4K12).

BRD-9853 showed minimal activity in this cell line. BRD-4097 was thenegative control. This is a benzamide with minimal HDAC inhibitoryactivity.

Standard western blotting methods were also used to measure the effectsof HDAC inhibitors on histone acetylation marks in HeLa cell lysate.Quantification of western blots in HeLa cells and the effect of compoundtreatment on the levels of H4K12 acetylation is shown in FIG. 11.Relative to the DMSO control, varying degrees of acetylation wereobserved. HDAC1,2 and HDAC1,2,3 selective compounds were found to beeffective at increasing the acetylation at the H4K12 loci.

Example 5 Functional Measures of BRD-6929 Cellular HDAC Activity

FIG. 12 demonstrates western blots of primary striatal cells isolatedfrom mouse brain that have been treated with HDAC inhibitors. Two setsof data with 3 independent samples/set are presented. Histogramsrepresenting the quantification of westerns are also shown. Relative toDMSO controls, BRD-6929 has a significant effect on the acetylationlevels of histone locus H4K12. BRD-6929 treatment results in a 5-10 foldincrease at 1 and 10 uM. BRD-6929 is an HDAC 1,2 selective compound, andhas 200× selectivity for HDAC1,2 vs. HDAC3. This demonstrates that anHDAC1,2 selective compound can effectively increase acetylation marksassociated with HDAC2 inhibition and memory, H4K12. In this case thedata was compared to controls: SAHA and BRD-3696 (CI-994). An HDAC1,2selective compound is as effective at increasing acetylation as anHDAC1,2,3 inhibitor and a pan inhibitor (i.e. SAHA). Inhibiting HDAC1,2is sufficient to effect increased acetylation at this histone locus.

FIG. 13 shows histograms representing the quantification of western gelanalysis examining additional acetylation marks in primary striatalcells. Four compounds were tested including CI-994 (BRD-3696) and SAHA.Relative to DMSO controls, BRD-6929 and BRD-5298 have significantlyincreased tetra-acetylated H4. Both compounds also show a trend towardincreasing tetra-acetylated H2B. BRD-6929 and BRD-5298 treatment resultsin a 2-5 fold increase in both marks at 1 and 10 uM. This datademonstrates that HDAC 1,2 specific compounds (BRD-6929, 5298) areeffective in increasing a specific acetylation associated with theinhibition of HDAC2 and learning and memory.

Example 6 In Vitro Data with Brd-6929 in Neuronal Cell Lines(Immunofluorescent Analysis)

Materials and Methods:

Day 1:

1) Compounds were pin transferred from 384-well plates (Abgene) using a185 nl pin tool using a no touch bottom protocol.

Day 2: After ˜24 hour compound treatment—

1) Media was aspirated using a plate washer (Tecan) protocol that leaves˜5 ul residual volume and without touching the bottom of plates); oralternatively, wells were gently aspirated to remove media with12-channel aspirator wand.

2) A multichannel pipet or use liquid handling system (e.g. Combi,standard tubing; slow speed) was used to add 75 ul formaldehyde (4% inPBS) and wells incubated 10 min at room temperature.

3) Formaldehyde was aspirated and cells rinsed 3 times with 100 ul PBS;

4) PBS was aspirated and 100 ul blocking/permeablization buffer (0.1%Triton-X100, 2% BSA, in PBS) added and wells incubate 1 hour at roomtemperature.

5) Blocking buffer was aspirated and 50 ul primary antibody diluted1:500 in blocking buffer was added and wells incubated overnight at 4degrees.

Day 3:

1) Primary antibody was aspirated and wells rinsed 3 times with 100 ulblocking buffer

2) 50 ul of secondary antibody diluted 1:500 and with Hoeschst (1:1000from 10 mg/mL (16 mM) stock) added and wells incubated 1.5 hours at roomtemperature covered in foil to prevent photobleaching.

3) Wells were rinsed 3 times with 100 ul PBS, and a 100 uls of PBS addedand the plates, sealed

4) Plates were then read on Acumen/IX Micro

5) Plates were stored at 4 degrees.

Results:

BRD-6929 at 1 and 10 uM does not cause an increase or decrease inoverall cell number after 6 h incubation in brain region specificprimary cultures (cortex and striatum). BRD-6929 at 10 uM causes anincrease in H4K12 acetylation after 6 h incubation in brain regionspecific primary cultures (striatum) (FIG. 14). BRD-6929 and BRD-5298(HDAC1,2 selective inhibitors) at 1 and 10 uM cause a significantincrease in H2B acetylation after 6 h incubation in primary neuronalcell cultures (FIGS. 15, 16). This demonstrates that HDAC 1,2 selectivecompounds are effective in increasing the acetylation at the specifichistone locus H2B. Increased acetylation of this histone locus isassociated with the inhibition or modulation of HDAC2 and learning andmemory. To our knowledge there are no reports of compounds with thisHDAC inhibitory selectivity eliciting these specific marks in thisspecific cell type.

Example 7 Concentration-Time Curve of BRD-6929 in Plasma and Brain

FIG. 17 represents a summary of the pharmacokinetic data after a singledose of 45 mg/kg BRD-6929 administered systemically via intraperitonealinjection. The concentration time curves for BRD-6929 in the plasma andbrain of C-57 mice from 5 min to 24 h are shown. This data demonstratesthat BRD-6929 crosses the blood-brain barrier and achievesconcentrations in excess of its HDAC 1 and 2 IC50 in whole brain. Thebrain C_(max)(0.83 uM) and the AUC (3.9 uM) levels are well aboveeffective in vitro concentrations necessary for enzymatic inhibition.

Example 8 Increase in Acetylation Marks in Brain Specific RegionsRelated to Learning and Memory after Acute Dosing in Mice

The experimental protocol for acute treatment with BRD-6929 and thecorresponding effects on histone acetylation in brain specific regionsof adult male C57BL/6J mice is shown in FIG. 18. Crude Protein LysisProtocol for western blot analysis of specific brain sections.

1. For dissected, frozen brain tissue:

a. On ice, thaw frozen tissue and immediately homogenize carefully in250 uL of ice-cold Suspension Buffer.

(100 uL was used for tissue approx. 2-3 mm3; adjust as needed)

1.5 mL disposable pestles (Fisher cat #03-392-100)

b. As soon as possible, add an equal volume of 2×SDS gel-loading buffer,pipetting up and down to mix.

2. Place the sample at 95° C. for 5 min.

3. Shear viscous chromosomal DNA by smoothly passaging through 23-25gauge hypodermic needle (2-3×) or by sonicating briefly (Al used theneedle method and it worked fine). Avoid foaming/bubbles.

4. Centrifuge the sample at 10,000 g for 10 min at room temperature,transferring supernatant to fresh tube.

5. Aliquot sample as needed based on protein concentration.

Suspension Buffer:

0.1M NaCl, 0.01M TrisCl (pH 7.6), 0.001M EDTA (pH 8.0) (buffer to thispoint can be prepared ahead, room temp. storage) Just before use, add:1× phosphatase/protease inhibitor cocktail (ex. ThermoFisher “HALT,” cat#78440) 5 mM Sodium Butyrate (HDAC inhibitor).

2×SDS Gel-Loading Buffer:

100 mM TrisC1 (pH 6.8), 4% SDS, 20% glycerol (buffer to this point canbe prepared ahead, room temp. storage) Just before use, add: 200 mMdithiothreitol (from 1M stock) 5 mM Sodium Butyrate (1-1DAC inhibitor).

Results: BRD-6929 causes a significant increase in the levels oftetra-acetylated H2B in the cortex of mice (FIG. 19). This demonstratesthat BRD-6929 is a functional inhibitor of HDACs in the cortex after asingle dose given systemically. BRD-6929 causes a 1.5-2 fold increase inthe acetylation levels for H2BK5 (FIG. 20). This acetylation mark hasbeen associated with increased learning and memory. These experimentsdemonstrate that BRD-6929, an HDAC 1,2 selective inhibitor, has enteredthe brain, and the nucleus of cells located in specific brain regionsassociated with learning and memory. Moreover, BRD-6929 causes anincrease in specific acetylation marks which have also been associatedwith learning and memory effects. To our knowledge, it has not beendemonstrated that a compound with this high level of HDAC 1,2 selectiveinhibition is efficacious in increasing acetylation levels in the brain.

Example 9 Increase in Acetylation Marks in Whole Brain after ChronicAdministration of BRD-6929

Western gel analysis demonstrated that even after chronic administrationof BRD-6929, every day for 10 days, BRD-6929 can still exert an effecton acetylation levels in the brains of mice. The western blot showed anincrease in tetra-acetylated H2B relative to the vehicle control (FIG.21). This demonstrates that an HDAC 1,2 selective compound caneffectively increase acetylation levels of specific acetylation marks(tetra-acetylated H2B) in the brain after chronic injection.

Example 10 Behavioral Data in Mice: Phenotypes that Correspond toImproved Memory and Cognition

C57/BL6 WT mice were injected with vehicle or BRD-6929 for 10 days. Onday 11, mice were trained in contextual fear conditioning paradigm(Training consisted of a 3 min exposure of mice to the conditioning box(context, TSE) followed by a foot shock (2 sec, 0.8 mA, constantcurrent). One hour after training, mice were injected with BRD-6929 orvehicle. On day 12, mice were returned to the training box and thefreezing behavior were monitored and recorded.

Result: A 45 mg/kg dose of BRD-6929 given every day for 10 days improvedthe memory of mice in a contextual fear conditioning paradigm asmeasured by % time freezing (FIG. 22). To our knowledge, this effect hasnot been reported previously for an HDAC1,2 selective compound or forthis class of compounds under any conditions. It was quite unexpectedthat this HDAC 1,2, selective inhibitor would be efficacious.

Example 11 Synthesis of(E)-3-(4-((2-acetamidoethylamino)methyl)phenyl)-N-(2-amino-5-(thiophen-2-yl)phenyl)acrylamide(BRD-9460)

A mixture of ethyl acetate (3.0 g, 34.1 mmol, 1.0 eq) andethylenediamine (6.14 g, 102 mmol, 3.0 eq) was stirred at roomtemperature for 4 days. The reaction mixture was then concentrated invacuo. The product was purified by flash chromatography (silica gel, 1%ammonia/49% CH₂Cl₂/50% MeOH) to afford the desired product as a yellowoil (2.0 g, 57% yield).

A mixture of 4-bromobenzaldehyde (9.25 g, 50.0 mmol, 1.0 eq), tert-butylacrylate (8.01 g, 62.5 mmom, 1.25 eq), triethylamine (10.12 g, 100 mmol,2.0 eq), triacetoxylpalladium (0.14 g, 0.5 mmol, 0.01 eq) andtri-o-tolylphosphine (0.609 g, 2.0 mmol, 0.04 eq) was heated at 100° C.for 2 h under nitrogen atmosphere. The reaction mixture was then dilutedwith water and extracted with ethyl acetate. The aqueous layer wasadjusted to pH˜3 with a 1M aqueous solution of HCl. The product wasextracted with ethyl acetate. The combined organic layer were filtered,dried over sodium sulfate and concentrated in vacuo to give the desiredproduct as a yellow solid (10.3 g, 89% yield).

A mixture of tert-butyl cinnamate (10.3 g, 44.3 mmol) in trifluoroacetic(100 mL) acid was stirred at room temperature overnight. The solventwere then removed by evaporation under reduced pressure. The obtainedyellow residue was dissolved in a saturated aqueous solution of sodiumcarbonate. The suspension was filtered and the filtrate was treated witha 3 M aqueous solution of HCl to give a white precipitate. Theprecipitate was then filtered off and dried to obtain the desiredproduct as a white solid (5.3 g, 67% yield).

To a stirred solution of 4-bromo-2-nitroaniline (50 g, 230.4 mmol, 1 eq)in DMF (800 mL) was added 60% NaH (6.1 g, 253.4 mmol, 1.1 eq) and(Boc)₂O (60.3 g, 276 mmol, 1.2 eq) in DMF (200 ml) at 0° C. The reactionmixture was stirred room temperature for 5 h. The reaction was thenpoured into ice-cold water and stirred for 1 h. The obtained solid wasfiltered and dried under reduced pressure. The crude material waspurified by column chromatography (silica gel, 1% EtOAc/hexanes) to givethe desired product (41.0 g, 56% yield).

To a stirred solution of compound tert-butyl4-bromo-2-nitrophenylcarbamate (1 g, 3.15 mmol, 1 eq) in DME (7 mL) wasadded thiophen-2-ylboronic acid (0.48 g, 3.78 mmol, 1.2 eq), Na₂CO₃ (1.0g, 9.46 mmol, 3.0 eq), tetrakis(triphenylphosphine)palladium(0) (0.36 g,0.31 mmol, 0.1 eq) and water (3 mL). The reaction mixture was heated at90° C. for 18 h. The reaction was diluted with EtOAc and water. Theorganic layer was separated, dried over sodium sulfate, filtered andconcentrated. The crude material was purified by column chromatography(silica gel, 10% EtOAc/hexanes) to afford the desired product (0.51 g,50% yield).

To a solution of tert-butyl 2-nitro-4-(thiophen-2-yl)phenylcarbamate(12.0 g, 37.5 mmol, 1 eq) in methanol (200 mL) was added hydrazinemonohydrate (80 mL) and iron (III) chloride (0.37 g, 2.24 mmol, 0.06eq). The reaction was stirred 80° C. for 1 h. The reaction was thenfiltered hot over celite and concentrated under reduced pressure. Theobtained residue was diluted with water (500 mL) and stirred well. Theobtained solid was filtered washed with water then hexanes and dried(10.5 g, 97% yield).

A mixture of (E)-3-(4-formylphenyl)acrylic acid (1.0 g, 5.68 mmol, 1.0eq), tert-butyl 2-amino-4-(thiophen-2-yl)phenylcarbamate (1.48 g, 5.11mmol, 0.9 eq) in THF was treated with HATU (2.2 g, 9.13 mmol, 1.0 eq)and DIPEA (1.8 g, 25.3 mmol, 4.46 eq). The resulting mixture was stirredat room temperature for 20 h. The solvents were removed by evaporation.The residue was diluted with ethyl acetate and washed with water, thenbrine. The organic layer was separated, dried over Na₂SO₄ andconcentrated under reduced pressure. The product was purified by columnchromatography (silica gel, 50% Petroleum ether/50% CH₂Cl₂) to afford acrude product as a yellow solid (2.2 g, 86% yield).

To a mixture of (E)-tert-butyl2-(3-(4-formylphenyl)acrylamido)-4-(thiophen-2-yl)phenylcarbamate (0.15g, 0.33 mmol, 1.0 eq) and N-(2-aminoethyl)acetamide (0.07 g, 0.67 mmol,2.0 eq) in dichloroethane was added NaBH(OAc)₃ (0.43 g, 2.01 mmol, 6.0eq). The reaction was stirred at room temperature for 20 h. The reactionwas concentrated, and ethyl acetate (30 mL) was added. The organicsolution was washed with a saturated aqueous solution of sodiumbicarbonate (10 mL), then brine (10 mL), the organic layer wasconcentrated in vacuo, the residue was purified by prep TLC (5%MeOH/CH₂Cl₂) to afford the desired product as a white solid (0.04 g, 22%yield).

A mixture of (E)-tert-butyl2-(3-(4-((2-acetamidoethylamino)methyl)phenyl)acrylamido)-4-(thiophen-2-yl)phenylcarbamate(0.04 g, 0.08 mmol) in dichloromethane (4 mL) was treated withtrifluoroacetic acid (1 mL) and stirred at room temperature for 1 h. Thereaction mixture was quenched with a saturated aqueous solution ofbicarbonate and extracted with ethyl acetate. The organic layer wasseparated, dried over sodium sulfate, filtered and concentrated. Theobtained yellow solid was washed with DCM/Hexane and dried under reducedpressure (0.02 g, 55.4% yield). ESI+ MS: m/z (rel intensity) 435 (100,M+H), ¹H NMR (500 MHz, d⁶-DMSO): δ 9.45 (s, 1H), 7.87-7.78 (m, 1H), 7.70(s, 1H), 7.63-7.52 (m, 3H), 7.45-7.32 (m, 3H), 7.28-7.20 (m, 2H),7.08-7.02 (m, 1H), 6.89 (d, J=15.5 Hz, 1H), 6.79 (d, J=8 Hz, 1H), 5.22(s, 2H), 3.74 (s, 2H), 3.14 (d, J=6 Hz, 2H), 2.60-2.42 (m, 2H), 1.79 (s,3H).

Synthesis ofN1-(2-amino-5-(pyridin-3-yl)phenyl)-N4-(2-(4-methylpiperazin-1-yl)ethyl)terephthalamide(BRD-6551)

A mixture of tert-butyl 2-amino-4-(pyridin-3-yl)phenylcarbamate (0.50 g,1.74 mmol, 1 eq) [which was prepared in a similar manner to tert-butyl2-amino-4-(thiophen-2-yl)phenylcarbamate], 4-(methoxycarbonyl)benzoicacid (0.47 g, 2.62 mmol, 1.5 eq), and BOP (1.4 g, 3.16 mmol, 1.8 eq) inpyridine (5 mL) was stirred at room temperature for 20 h. The solventwas removed by evaporation. The residue was then diluted with asaturated aqueous solution of sodium bicarbonate. The obtained solid wasfiltered. The crude product purified by column chromatography (silicagel, 2% MeOH/CH₂Cl₂) to give the desired product (0.68 g, 87% yield).

A solution of methyl4-(2-(tert-butoxycarbonylamino)-5-(pyridin-3-yl)phenylcarbamoyl)benzoate(0.68 g, 1.52 mmol, 1 eq) in THF (10 mL) was treated with a solution oflithium hydroxide (0.18 g, 7.60 mmol, 5.0 eq) in water (10 mL). Thereaction was stirred at room temperature 2 h. The reaction wasconcentrated then diluted with water and adjusted to pH˜3 with citricacid. The obtained solid was filtered and used directly in the nextreaction (0.61 g, 93% crude yield).

A mixture of4-(2-(tert-butoxycarbonylamino)-5-(pyridin-3-yl)phenylcarbamoyl)benzoicacid (0.20 g, 0.46 mmol, 1.0 eq), 2-(4-methylpiperazin-1-yl)ethanamine(0.13 g, 0.92 mmol, 2.0 eq) in DMF (4 mL) was treated with HATU (0.35 g,0.92 mmol, 2.0 eq) and DIPEA (0.20 mL, 1.15 mmol, 2.5 eq). The reactionwas stirred at room temperature for 20 h. Water was added. The obtainedsolid was filtered and dried. The crude product was purified by columnchromatography (silica gel, 30% EtOAc/hexanes) to afford the desiredproduct (0.19 g, 77% yield).

A 4M solution of HCl in 1,4-dioxane (2 mL) was added to a stirredsolution of tert-butyl2-(4-(2-(4-methylpiperazin-1-yl)ethylcarbamoyl)benzamido)-4-(pyridin-3-yl)phenylcarbamate(0.10 g, 0.18 mmol, 1 eq) in methanol (2 mL) at 0° C. The reaction wasthen warmed to room temperature and stirred for 2 h. The solvents wereremoved by evaporation and a saturated aqueous solution of sodiumbicarbonate was added. The obtained solid was filtered and dried undervacuum to get the desired compound (0.05 g, 60% yield). ESI+ MS: m/z(rel intensity) 459 (96.6, M+H), ¹H NMR (500 MHz, d6-DMSO): δ 9.84 (s,1H), 8.79 (d, J=1.5 Hz, 1H), 8.53 (t, J=5.5 Hz, 1H), 8.44 (dd, J=4.0,1.5 Hz, 1H), 8.08 (d, J=8.5 Hz, 2H), 7.95 (d, J=8.5 Hz, 3H), 7.58 (s,1H), 7.42-7.38 (m, 2H), 6.90 (d, J=8.5 Hz, 1H), 5.23 (bs, 1H), 3.42-3.36(m, 2H), 2.55-2.20 (m, 10H), 2.15 (s, 3H).

One skilled in the art will recognize that other compounds describedbelow can be prepared in a similar manner to the procedures describedabove.

N1-(2-amino-5-(thiophen-2-yl)phenyl)-N4-(2-(4-methylpiperazin-1-yl)ethyl)terephthalamide(BRD-5298) can be prepared by substituting pyridin-3-ylboronic acid withthiophen-2-ylboronic acid. ESI+ MS: m/z (rel intensity) 464 (98.27,M+H).

The following four compounds can be prepared by substituting2-(4-methylpiperazin-1-yl)ethanamine with 2-phenylethanamine andutilizing the appropriate benzoic acid.

N1-(2-aminophenyl)-N4-phenethylterephthalamide (BRD-1783), ESI+ MS: m/z(rel intensity) 359 (100, M).

N1-(2-amino-5-(thiophen-2-yl)phenyl)-N4-phenethylterephthalamide(BRD-8451), ESI+ MS: m/z (rel intensity) 442 (99.34, M+H).

N1-(2-amino-5-(pyridin-3-yl)phenyl)-N4-phenethylterephthalamide(BRD-0984), ESI+ MS: m/z (rel intensity) 437 (95.85, M+H).

N1-(2-amino-5-(thiophen-2-yl)phenyl)-N4-(2-(pyridin-4-yl)ethyl)terephthalamide(BRD-6597) can be prepared by substituting2-(4-methylpiperazin-1-yl)ethanamine with 2-(pyridin-4-yl)ethanamine.ESI+ MS: m/z (rel intensity) 443 (97.68, M+H).

Synthesis of(E)-N-(2-amino-5-(thiophen-2-yl)phenyl)-4-(3-oxo-3-(2-(pyridin-2-yl)ethylamino)prop-1-enyl)benzamide(BRD-9853)

A mixture of (E)-3-(4-(methoxycarbonyl)phenyl)acrylic acid (0.25 g, 1.21mmol, 1.0 eq), 2-(pyridin-2-yl)ethanamine (0.30 g, 2.42, 2.0 eq), HATU(0.46 g, 1.91, 1.57 eq), DIPEA (0.31 g, 4.36 mmol, 3.6 eq) in THF (10mL) was stirred at room temperature for 20 h. The reaction wasconcentrated and ethyl acetate (30 mL) was added. The organic layer waswashed with water (20 mL), dried over magnesium sulfate, filtered andconcentrated. The product was purified by column chromatography (silicagel, 10% MeOH/CH₂Cl₂) to provide the desired compound (0.20 g, 52%yield). ESI+ MS: m/z (rel intensity) 311 (98.7, M+H).

To a solution of (E)-methyl4-(3-oxo-3-(2-(pyridin-2-yl)ethylamino)prop-1-enyl)benzoate (0.20 g,0.64 mmol, 1.0 eq) in THF (3 mL) was added a solution of LiOH (0.05 g,1.93 mmol, 3.0 eq) in water (3 mL). The reaction was stirred at roomtemperature for 20 h. The reaction was then concentrated and dilutedwith water (5 mL). The solution was acidified with a 1N aqueous solutionof HCl to pH˜2. The precipitate formed was filtered and rinsed withwater (3 mL) to afford a white solid (0.15 g, 79% crude yield). ESI+ MS:m/z (rel intensity) 297 (63.8, M+H).

A mixture of(E)-4-(3-oxo-3-(2-(pyridin-2-yl)ethylamino)prop-1-enyl)benzoic acid(0.10 g, 0.34 mmol, 1.0 eq), tert-butyl2-amino-4-(thiophen-2-yl)phenylcarbamate (0.19 g, 0.68 mmol, 2.0 eq),HATU (0.46 g, 1.91 mmol, 5.6 eq) and DIPEA (0.31 g, 4.36 mmol, 12.9 eq)in THF (10 mL) was stirred at room temperature for 20 h. The reactionwas concentrated and ethyl acetate (30 mL) was added. The solution waswashed with water (20 mL). The combined organic layers were dried oversodium sulfate, filtered and concentrated. The product was purified bycolumn chromatography (silica gel, 6% MeOH/CH₂Cl₂) to provide the targetcompound (0.11 g, 56% yield). ESI+ MS: m/z (rel intensity) 569 (98.5,M+H).

To a solution of (E)-tert-butyl2-(4-(3-oxo-3-(2-(pyridin-2-yl)ethylamino)prop-1-enyl)benzamido)-4-(thiophen-2-yl)phenylcarbamate(0.11 g, 0.19 mmol, 1.0 eq) in dichloromethane (4 mL) was addedtrifluoroacetic acid (1.5 mL). The reaction was stirred at roomtemperature for 1 h and concentrated. The residue was dissolved in ethylacetate (20 mL), washed with a saturated aqueous solution of sodiumbicarbonate (10 mL), water (10 mL). The organic layer was dried oversodium sulfate, filtered and concentrated. The residue was washed withether (2 mL) to give a yellow solid (0.07 g, 75% yield). ESI+ MS: m/z(rel intensity) 469 (97.9, M+H), ESI+ MS: m/z (rel intensity) 469 (97.9,M+H), ¹H NMR (500 MHz, d⁶-DMSO): δ 9.79 (s, 1H), 8.52 (d, J=4 Hz, 1H),8.30-8.23 (m, 1H), 8.034 (d, J=7.5 Hz, 2H), 7.75-7.65 (m, 3H), 7.48 (d,J=15 Hz, 2H), 7.36 (d, J=5 Hz, 1H), 7.30 (t, J=8.5 Hz, 2H), 7.26-7.20(m, 2H), 7.05 (t, J=4.5 Hz, 2H), 6.81 (d, J=8.5 Hz, 1H), 6.74 (d, J=15.5Hz, 1H), 5.19 (s, 2H), 3.60-3.52 (m, 2H), 2.95 (t, J=7 Hz, 2H).

Synthesis of(E)-3-(3-(2-amino-5-(thiophen-2-yl)phenylamino)-3-oxoprop-1-enyl)benzamide(BRD-3636)

A mixture of methyl 3-bromobenzoate (10.8 g, 50.2 mmol, 1.0 eq), t-butylacrylate (8.05 g, 62.8 mmol, 1.25 eq), triethylamine (10.16 g, 100 mmol,2.0 eq), triacetoxylpalladium (0.14 g, 0.50 mmol, 0.01 eq) andtri-o-tolylphosphine (0.61 g, 2.0 mmol, 0.04 eq) was heated at 100° C.for 2 h under nitrogen atmosphere. The reaction mixture was diluted withwater. The product was extracted with ethyl acetate. The organic phasewas adjusted to pH˜3 with a 1M aqueous solution of HCl. The organiclayer was separated, dried over sodium sulfate, filtered andconcentrated in vacuo to give a yellow solid (11 g, 84% yield).

A mixture (E)-methyl 3-(3-tert-butoxy-3-oxoprop-1-enyl)benzoate (12.0 g,45.7 mmol) in TFA (100 mL) was stirred at room temperature for 20 h. Thesolvent was removed under reduced pressure. The residue obtained waswashed with ethyl acetate to give a white solid (8.5 g, 90% yield).

A mixture of (E)-3-(3-(methoxycarbonyl)phenyl)acrylic acid (5.56 g, 27mmol, 1.5 eq), tert-butyl 2-amino-4-(thiophen-2-yl)phenylcarbamate (5.22g, 17.98 mmol, 1.0 eq), HATU (10.30, 42.7 mmol, 2.37 eq) and DIPEA (6.96g, 98 mmol, 5.45 eq) in THF (80 ml) was stirred at room temperature for20 h. The reaction was then concentrated. The residue was diluted withethyl acetate and washed with water, then brine. The organic layer wasseparated, dried over sodium sulfate, filtered and concentrated underreduced pressure. The product was purified by column chromatography(silica gel, 50% PE/CH₂Cl₂) to afford a yellow solid (6.0 g, 64.9%yield).

To a solution of (E)-methyl3-(3-(2-(tert-butoxycarbonylamino)-5-(thiophen-2-yl)phenylamino)-3-oxoprop-1-enyl)benzoate(5.5 g, 11.49 mmol, 1.0 eq) in THF (60 mL) was added a solution of LiOH(0.69 g, 28.7 mmol, 2.5 eq) in water (60 mL). The reaction was stirredat room temperature for 20 h. The reaction was extracted with ethylacetate. The aqueous layer was separated and acidified with a 1N aqueoussolution of HCl to pH˜2. The precipitate formed was filtered and rinsedsubsequently with water (200 mL), then methanol (100 mL) to afford awhite solid (4.2 g, 79% yield).

A mixture of ammonia hydrochloride (0.02 g, 0.43 mmol, 2.0 eq),(E)-3-(3-(2-(tert-butoxycarbonylamino)-5-(thiophen-2-yl)phenylamino)-3-oxoprop-1-enyl)benzoicacid (0.10 g, 0.21 mmol, 1.0 eq), HATU (0.08 g, 0.32 mmol, 1.5 eq), HOBt(0.043 g, 0.32 mmol, 1.5 eq) and DIPEA (0.11 g, 0.86 mmol, 4.0 eq) inTHF (10 mL) was stirred at room temperature for 20 h. The reaction wasthen concentrated. The residue was diluted with ethyl acetate and washedwith water, then brine. The organic layer was separated, dried oversodium sulfate, filtered and concentrated under reduced pressure. Theproduct was purified by column chromatography (silica gel, 10%MeOH/CH₂Cl₂) to afford the desired product (0.08 g, 80% yield).

A solution of (E)-tert-butyl2-(3-(3-carbamoylphenyl)acrylamido)-4-(thiophen-2-yl)phenylcarbamate(0.08 g, 0.17 mmol, 1.0 eq) in CH₂Cl₂ was treated with trifluoroaceticacid (1 mL). The solution was stirred at room temperature for 1 h. Thereaction was then concentrated. The residue was dissolved in ethylacetate (20 mL). The solution was washed with a saturated aqueoussolution of sodium bicarbonate (10 mL), then water (10 mL). The combinedorganic layers were dried over sodium sulfate, filtered andconcentrated. The product was washed with ether (2 mL) to give thetarget compound (0.05 g, 73% yield). ESI+ MS: m/z (rel intensity) 364(92.63, M+H), ¹H NMR (500 MHz, d⁶-DMSO): δ 9.48 (s, 1H), 8.18 (s, 1H),8.09 (s, 1H), 7.90 (d, J=8 Hz, 1H), 7.80-7.70 (m, 2H), 7.62 (d, J=16 Hz,1H), 7.54 (t, J=8 Hz, 1H), 7.49 (s, 1H), 7.36 (d, J=5 Hz, 1H), 7.29-7.19(m, 2H), 7.10-6.59 (m, 2H), 6.79 (d, J=8 Hz, 1H), 5.24 (s, 2H).

Synthesis of 4-acetamido-N-(4-amino-2′-methylbiphenyl-3-yl)benzamide(BRD-4029)

A mixture of tert-butyl 4-bromo-2-nitrophenylcarbamate (0.20 g, 0.62mmol), o-tolylboronic acid (0.10 g, 0.74 mmol), sodium carbonate (0.20g, 0.93 mmol) and Pd(PPh₃)₄ (50 mg, 0.04 mmol) in DME/H₂O (2:1, 5 mL)was heated to 110° C. under argon atmosphere. After vigorously stirringfor 20 h, water was added. The product was extracted with ethyl acetate.The combined organic layers were washed with water, dried over sodiumsulfate, filtered and concentrated. The residue was purified bychromatography (silica gel, 10% EtOAc/PE) to give the desired product asyellow solid (0.13 mg, 95% yield). ¹HNMR (400 MHz, DMSO-d6): 1.46 (s,9H), 2.25 (s, 3H), 7.26-7.32 (m, 4H), 7.66-7.70 (m, 2H), 7.85 (d, J=1.6Hz, 1H), 9.67 (s, 1H).

A solution of tert-butyl 2′-methyl-3-nitrobiphenyl-4-ylcarbamate (0.13g, 0.58 mmol), Pd/C (10%, 0.06 g) in MeOH (5 mL) was vigorously stirredfor 16 h under hydrogen atmosphere. The reaction was filtered throughCelite. The filtrate was concentrated. The product was purified bycolumn chromatography (silica gel, 2.5% EtOAc/PE) to give the desiredproduct as yellow solid (0.161 g, 92% yield). ¹HNMR (400 MHz, d⁶-DMSO):1.47 (s, 9H), 2.22 (s, 3H), 4.90 (s, 2H), 6.48 (dd, J=8.0, 1.6 Hz, 1H),6.64 (d, J=1.6 Hz, 1H), 7.11-7.13 (m, 1H), 7.19-7.25 (m, 4H), 8.34 (s,1H).

A solution of tert-butyl 3-amino-2′-methylbiphenyl-4-ylcarbamate (0.15g, 0.508 mmol, 1.0 eq), 4-acetamidobenzoic acid (0.56 g, 3.06 mmol, 6eq), DIPEA (0.7 mL, 4.08 mmol, 8 eq) and HATU (0.39 g, 1.14 mmol, 2 eq)in DMF (5 mL) was stirred for 16 h under argon atmosphere. Water wasadded and the product was extracted with EtOAc. The combined organiclayers were dried over sodium sulfate, filtered and concentrated. Theproduct was purified by column chromatography (silica gel, 10% EtOAc/PE)to give the desired product as yellow solid (0.10 g, 42% yield). ¹HNMR(400 MHz, d⁶-DMSO): δ 1.47 (s, 9H), 2.08 (s, 3H), 2.28 (s, 3H), 7.16-7.3(m, 5H), 7.53 (d, J=1.2 Hz, 1H), 7.57 (d, J=8.4 Hz, 1H), 7.72 (d, J=8.8Hz, 2H), 7.90-7.92 (d, J=8.8 Hz, 1H), 8.80 (s, 1H), 9.80 (s, 1H), 10.24(s, 1H).

A solution of tert-butyl3-(4-acetamidobenzamido)-2′-methylbiphenyl-4-ylcarbamate (0.06 g, 0.124mmol) in CH₂Cl₂ (1.5 mL) was treated with TFA (0.7 mL) at 0° C. Afterthe reaction mixture was stirred at 0° C. for 2 h, the reaction wasdiluted with ethyl acetate and washed with a saturated solution ofsodium bicarbonate. The combined organic layers were dried over sodiumsulfate, filtered and concentrated to give the desired product (0.03 g,47% yield). ¹HNMR (400 MHz, d⁶-DMSO): δ 2.08 (s, 3H), 2.27 (s, 3H), 5.00(s, 2H), 6.84 (d, J=8.4 Hz, 1H), 6.96 (d, J=8.0, 2.0 Hz, 1H), 7.16-7.25(m, 5H), 7.69 (d, J=8.8 Hz, 2H), 7.94 (d, J=8.8 Hz, 2H), 9.60 (s, 1H),10.20 (s, 1H). MS: m/z (360, [M+H]⁺; 382, [M+Na]⁺).

One skilled in the art will recognize that other compounds describedbelow can be prepared in a similar manner to the procedures describedabove.

4-acetamido-N-(2-amino-5-(pyridin-3-yl)phenyl)benzamide (ORD-9773) canbe prepared by substituting o-tolylboronic acid with pyridin-3-ylboronicacid. ESI+ MS: m/z (rel intensity) 369 (96.2, M+Na).

4-acetamido-N-(2-amino-5-(thiophen-2-yl)phenyl)benzamide (BRD-6929) canbe prepared by substituting o-tolylboronic acid withthiophen-2-ylboronic acid. ESI+ MS: m/z (rel intensity) 382 (96.2,M+Na).

Synthesis of N-(2-amino-5-(thiophen-2-yl)phenyl)-4-sulfamoylbenzamide(BRD-7726)

A solution of tert-butyl 2-amino-4-(thiophen-2-yl)phenylcarbamate (0.30g, 1.03 mmol, 1 eq), 4-sulfamoylbenzoic acid (0.42 g, 2.06 mmol, 2 eq),HATU (780 mg, 2.06 mmol, 2.0 eq.), and DIPEA (0.45 mL, 2.58 mmol, 2.5eq) in DMF (5 mL) was stirred for 15 h at room temperature. The reactionwas quenched with a saturated solution of sodium bicarbonate. The solidobtained was filtered and dried under reduced pressure. The product waspurified by column chromatography (silica gel, 3% MeOH/CH₂Cl₂) to affordthe desired product (0.25 g, 51% yield).

To a stirred solution of tert-butyl2-(4-sulfamoylbenzamido)-4-(thiophen-2-yl)phenylcarbamate (0.15 g, 0.32mmol, 1 eq) in CH₂Cl₂ (2 mL) was added TFA (1 mL) at 0° C. The reactionwas stirred at room temperature for 2 h. The reaction was thenconcentrated. The residue was dissolved in EtOAc. The solution waswashed with a saturated solution of sodium bicarbonate. The combinedorganic layers were dried over sodium sulfate, filtered andconcentrated. The product was purified by column chromatography (silicagel, 3% MeOH/CH₂Cl₂) to afford the desired product (0.03 g, 24.9%yield). ESI+ MS: m/z (rel intensity) 374 (98.32, M+H), ¹H NMR (500 MHz,d⁶-DMSO): δ 8.15 (d, J=8.5 Hz, 2H), 7.94 (d, J=8 Hz, 2H), 7.49 (s, 1H),7.36 (d, J=4.5 Hz, 1H), 7.31 (dd, J=8.5, 2 Hz, 1H), 7.44 (d, J=2.5 Hz,1H), 7.05 (t, J=3.5 Hz, 1H), 6.81 (d, J=8.5 Hz, 1H), 5.21 (s, 2H).

Synthesis of 4-acetamido-N-(2-amino-5-phenethylphenyl)benzamide(BRD-7050)

To a stirred solution of 5-bromo-2-nitroaniline (4.0 g, 18.43 mmol, 1.0eq), 4-acetamidobenzoic acid (4.95 g, 27.6 mmol, 1.5 eq) and BOP (10.60g, 23.96 mmol, 1.3 eq) was added sodium hydride (2.96 g, 123.0 mmol, 6.7eq) portion-wise at 0° C. The reaction mixture was allowed to warm toroom temperature and stir for 60 h. The solvents were evaporated underreduced pressure. The residue was diluted with a saturated solution ofsodium bicarbonate. The obtained precipitate was filtered. The productwas purified by column chromatography (silica gel, 25% EtOAc/CH₂Cl₂) toafford the desired product (3.31 g, 40% yield).

A mixture of tert-butyl 2-(4-acetamidobenzamido)-4-bromophenylcarbamate(0.50 g, 1.11 mmol, 1.0 eq), (E)-styrylboronic acid (0.33 g, 2.23 mmol,2.0 eq), potassium carbonate (0.46 g, 3.335 mmol, 3.0 eq), Pd(PPh₃)₄(0.09 g, 0.08 mmol, 0.07 eq) and tritolyl phosphine (0.10 g, 0.33 mmol,0.3 eq) in DME/H₂O (30 mL) was heated to reflux for 20 h. The reactionmixture was diluted with water. The obtained solid was filtered. Thecrude product was purified by column chromatography (silica gel, 2%MeOH/CH₂Cl₂) to obtain pure product (0.30 g, 57% yield).

To a solution of (E)-tert-butyl2-(4-acetamidobenzamido)-4-styrylphenylcarbamate (0.15 g, 0.32 mmol, 1.0eq) in ethanol (10 mL) was added palladium on carbon (0.02 g, 0.23 mmol,0.7 eq). The reaction mixture was stirred under H₂ atmosphere for 20 h.The reaction mixture was filtered through celite, the solids were washedwith methanol. The reaction was then concentrated under reduced pressureto afford an off white solid (0.14 g, 93% crude yield) which was used inthe next step without further purification.

To a solution of tert-butyl2-(4-acetamidobenzamido)-4-phenethylphenylcarbamate (0.14 g, 0.29 mmol)in CH₂Cl₂ (3 mL) at 0° C. was added TFA (2 mL) dropwise. The reactionmixture was slowly warmed to room temperature and stirred for 2 h. Thesolvent was removed by evaporation under reduced pressure. The cruderesidue was diluted with water and quenched with a saturated aqueoussolution of sodium bicarbonate. The obtained solid was filtered, washedwith water and dried under vacuum to afford the desired product (0.08 h,68% yield). ESI+ MS: m/z (rel intensity) 374 (95.0, M+H).

Synthesis of(E)-3-(4-acetamidophenyl)-N-(2-amino-5-ethynylphenyl)acrylamide(BRD-0063)

A mixture of N-(4-bromophenyl)acetamide (2.14 g, 10 mmol, 1.0 eq),tert-butyl acrylate (1.6 g, 13 mmol, 1.3 eq), diacetoxypalladium (0.05g, 0.2 mmol, 0.02 eq), P(o-tol)₃ (0.12 g, 0.4 mmol, 0.04 eq) intriethylamine (3 mL) was heated to 100° C. for 2 h under nitrogen. Thereaction was cooled to room temperature. Ethyl acetate (50 mL) wasadded. The organic layer was washed with water (2×20 mL). The combinedorganic layers were dried over sodium sulfate, filtered and concentratedto give the desired product (2.2 g, 76% crude yield) as a yellow solid.

(E)-tert-butyl 3-(4-acetamidophenyl)acrylate (2.2 g, 8.42 mmol) intrifluoroacetic acid (10 mL) was stirred at room temperature for 10 min.The solvent was removed by evaporation. The residue was dissolved inaqueous sodium carbonate (0.3N, 30 mL). The aqueous layer was washedwith ethyl acetate (2×20 ml), acidified to pH˜3 with a 1N aqueoussolution of hydrochloride acid. The product was extracted with ethylacetate (50 mL). The combined organic layers were dried over sodiumsulfate, filtered and concentrated to get the desired product (1.4 g,77% crude yield).

A mixture of tert-butyl 4-bromo-2-nitrophenylcarbamate (0.97 g, 3.08mmol, 1.0 eq), ethynyltrimethylsilane (0.45 g, 4.62 mmol, 1.5 eq),PdCl₂(PPh₃)₂ (0.11 g, 0.15 mmol, 0.05 eq) and CuI (0.04 g, 0.18 mmol,0.06 eq) in Et₃N (35 mL) was refluxed at 100° C. for 2 h. The reactionwas cooled to room temperature and the solvent was removed byevaporation. The residue was taken up in water (50 mL) and ethyl acetate(50 mL). The organic layer was dried over sodium sulfate, filtered andconcentrated to get the crude product (1.2 g, 116% crude yield) asyellow oil which was used without further purification in the next step.

A mixture of tert-butyl2-nitro-4-((trimethylsilyl)ethynyl)phenylcarbamate (1.2 g, 3.59 mmol,1.0 eq), SnCl₂.2H₂O (4.05 g, 17.94 mmol, 5.0 eq) and Et₃N (15 mL) inethanol (30 mL) was heated to 70° C. for 1 h. The reaction was thencooled to room temperature. The solvents were removed by evaporation.The residue was taken up in water (50 mL) and ethyl acetate (50 mL). Theorganic layer was dried over sodium sulfate, filtered and concentrated.The crude product was purified by column chromatography (silica gel, 25%EtOAc/PE) to afford the desired product (0.58 g, 53% yield) as yellowsolid.

A solution of tert-butyl2-amino-4-((trimethylsilyl)ethynyl)phenylcarbamate (0.20 g, 0.65 mmol,1.0 eq) in methanol (10 mL) was treated with K₂CO₃ (0.45 g, 3.28 mmol,5.0 eq). The reaction was stirred at room temperature for 30 min. Thesolvent was evaporated and the residue was taken up in water (20 mL) andethyl acetate (30 mL). The organic layer was dried over sodium sulfate,filtered and concentrated to get the desired product (0.14 g, 89% yield)which was used without further purification in the next step.

A mixture of (E)-3-(4-acetamidophenyl)acrylic acid (0.14 g, 0.70 mmol,1.2 eq), tert-butyl 2-amino-4-ethynylphenylcarbamate (0.13 g, 0.58 mmol,1.0 eq), HATU (0.26 g, 0.70 mmol, 1.2 eq) and DIPEA (0.23 g, 1.75 mmol,3.0 eq) in THF (10 mL) was stirred at room temperature for 20 h. Thesolvents were evaporated under reduced pressure. The residue was dilutedwith water (20 mL). The product was extracted with ethyl acetate (20 mL)twice. The combined organic layers were washed with brine, dried oversodium sulfate, filtered and concentrated. The product was purified bycolumn chromatography (silica gel, 6% MeOH/CH₂Cl₂) to afford the desiredproduct (0.12 g, 49% yield).

A solution of (E)-tert-butyl2-(3-(4-acetamidophenyl)acrylamido)-4-ethynylphenylcarbamate (0.06 g,0.14 mmol) in 1,4 dioxane (1 mL) at room temperature was treated with asolution of H₂SO₄ (0.10 g, 7.15 mmol, 50 eq) in 1,4 dioxane (1 mL). Theresulting mixture was stirred at room temperature for 2 h. The reactionmixture was quenched with a saturated aqueous solution of sodiumbicarbonate. The product was extracted with ethyl acetate. The combinedorganic layers was dried over sodium sulfate, filtered and concentrated.The product was purified by column chromatography (silica gel, 6%MeOH/CH₂Cl₂) to afford the desired product (0.01 g, 21% yield). ESI+ MS:m/z (rel intensity) 319 (94.13, M+H).

All references, patents and patent publications that are recited in thisapplication are incorporated in their entirety herein by reference.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

1. A method for enhancing a memory in a subject comprising administeringto the subject an HDAC2 inhibitor in an amount effective to enhance thememory in the subject, wherein the HDAC2 inhibitor is a selective HDAC2inhibitor. 2-7. (canceled)
 8. The method of claim 1, wherein a synapticnetwork in the subject is re-established.
 9. The method of claim 1,wherein the HDAC2 inhibitor is not an HDAC1 inhibitor.
 10. The method ofclaim 1, wherein the HDAC2 inhibitor is not an HDAC5, HDAC6, HDAC7 orHDAC10 inhibitor. 11-12. (canceled)
 13. The method of claim 1, whereinthe selective HDAC2 inhibitor is an HDAC2 RNAi such as a siRNA, shRNA,miRNA, dsRNA or ribozyme or variants thereof. 14-22. (canceled)
 23. Amethod for treating Alzheimer's disease comprising, administering to asubject having Alzheimer's disease an HDAC2 inhibitor in an amounteffective to treat Alzheimer's disease, wherein the HDAC2 inhibitor is aselective HDAC2 inhibitor.
 24. (canceled)
 25. The method claim 23wherein the HDAC2 inhibitor is a selective HDAC1/HDAC2 inhibitor. 26.The method of claim 25, wherein the HDAC2 inhibitor is a compound offormula (IV)

wherein R₁ and R₂ are independently selected from H, and—C(O)—C₁₋₆alkyl; R₃ is optionally substituted aryl, optionallysubstituted heteroaryl, or aryl-C₁₋₆alkylene.
 27. The method of claim26, wherein formula IV is


28. The method of claim 26, wherein formula IV is


29. The method of claim 26, wherein formula IV is


30. The method of claim 26, wherein formula IV is


31. The method of claim 23 wherein the HDAC2 inhibitor is a selectiveHDAC1/HDAC2/HDAC3 inhibitor.
 32. The method of claim 31, wherein theHDAC2 inhibitor is a compound of formula (VI)

wherein R₁ and R₂ are independently selected from H, substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₁₋₆alkyl,heterocyclyl, heteroaryl, aryl, and aryl-C₁₋₆alkylene.
 33. The method ofclaim 32, wherein formula VI is


34. The method of claim 23, wherein the HDAC2 inhibitor is a compound offormula (I)

wherein R₁ and R₂ are independently selected from H, substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₁₋₆alkyl,heterocyclyl, C₁₋₆alkylene, heteroaryl, heteroarylene, andheteroarylene-alkylene; and R₃ is aryl or heteroaryl.
 35. The method ofclaim 34, wherein formula I is


36. The method of claim 23, wherein the HDAC2 inhibitor is a compound offormula (II)

wherein R₁ and R₂ are independently selected from H, substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₁₋₆alkyl,heterocyclyl, C₁₋₆alkylene, heteroaryl, heteroarylene,heteroarylene-alkylene, arylene-alkylene; and heterocyclyl-alkyleneoptionally substituted; and R₃ is aryl or heteroaryl.
 37. The method ofclaim 36, wherein formula II is


38. The method of claim 36, wherein formula II is


39. The method of claim 36, wherein formula II is


40. The method of claim 36, wherein formula II is


41. The method of claim 36, wherein formula II is


42. The method of claim 23, wherein the HDAC2 inhibitor is a compound offormula (III)

wherein X is —C(O)—N(R₁)(R₂),C₁₋₆alkylene-N(H)—C₁₋₆alkylene-N(R₁)C(O)(R₂); or —N(R₁)C(O)R₂; R₁ and R₂are independently selected from H, and substituted or unsubstituted,branched or unbranched, cyclic or acyclic C₁₋₆alkyl; and R₃ is alkynyl,aryl, or heteroaryl.
 43. The method of claim 42, wherein formula III is


44. The method of claim 42, wherein formula III is


45. The method of claim 42, wherein formula III is


46. The method of claim 23, wherein the HDAC2 inhibitor is a compound offormula (V)

wherein R₁ and R₂ are independently selected from H, and substituted orunsubstituted, branched or unbranched, cyclic or acyclic C₁₋₆alkyl; andR₃ is aryl or heteroaryl.
 47. The method of claim 46, wherein formula Vis

48-51. (canceled)
 52. The method of claim 23 wherein the HDAC2 inhibitoris a selective HDAC1/HDAC2/HDAC10 inhibitor.
 53. The method of claim 23wherein the HDAC2 inhibitor is a selective HDAC1/HDAC2/HDAC3/HDAC10inhibitor.